System for encapsulating cells in three-dimensional culture compartments

Abstract

The invention relates to a system (1) for encapsulating cells, comprising at least two containers (21, 22), one of the containers comprising a solution of cells (S1) and the other of the containers comprising a solution capable of gelling (S2); an encapsulation device (3) connected to the containers via distributors (21a, 22a) and arranged to form cellular microcompartments (MC), the outer layer of which is the solution capable of gelling and the core of which is the solution of cells; a stiffening bath (4) arranged to collect the cellular microcompartments and to cause stiffening of the outer layer of each collected cellular microcompartment; characterized in that it comprises at least one member (22b) capable of electrically charging at least one of the solutions with a first electrical potential (V1), and in that the stiffening bath is electrically charged with a second electrical potential (V2).

Description

Description Title of the invention: System for encapsulating cells in three-dimensional culture compartments

[0001] The invention relates to the field of encapsulation of cells in three-dimensional cell culture compartments. More specifically, the invention relates to an encapsulation system for obtaining such compartments.

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

[0003] In these various applications, growing cells in large quantities represents a significant challenge. The research topics mentioned require a significant 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] An important advance in cell culture techniques is the introduction of three-dimensional culture systems. Three-dimensional cultures are indeed advantageously closer to natural in vivo systems, and can be used for many applications, particularly in the development of therapies. A particularly suitable technology is that described in application WO2018 / 096277, which consists of three-dimensional cellular microcompartments for the culture of stem cells. This document describes a cell encapsulation device comprising a microfluidic injector for forming cellular microcompartments in the form of drops, the outer layer of which is formed by a solution comprising alpha-Iginate and the core of which is formed by a solution of cells. These drops are collected in a calcium bath to stiffen their outer layer to form a shell.

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

[0006] In this device, the alginate solution entering the microfluidic injector is electrically charged, which generates an electrostatic force that facilitates the separation between the drops at the exit of the microfluidic injector, and prevents their fusion between the latter and the calcium bath.

[0007] However, as they are collected in the bath, the drops transfer their charges to the bath, which gradually becomes charged, which has the effect of repelling, by electrostatic force, the new drops that are collected by the bath. This can then lead to a loss of all or part of the drops produced as well as deterioration of the capsule shell or even an alteration of their shape. These various conformity defects thus significantly impair the production of microcompartments and thus go against large-scale industrial cell culture, and in particular of human pluripotent stem cells.

[0008] There is therefore a need for a three-dimensional cell culture system that can increase the yield and quality of cell culture.

[0009] The present invention is placed in this context and aims to meet this need.

[0010] For these purposes, the invention relates to a cell encapsulation system, the system comprising at least: a. two containers, one of the containers comprising a cell solution and the other of the containers comprising a solution capable of gelling, b. an encapsulation device provided with several inlets each connected to one of the containers via at least one distributor and arranged to form, at the outlet of the encapsulation device, from the solutions supplied by the distributor(s), cellular microcompartments whose outer layer is the solution capable of gelling and the core the cell solution, c. a stiffening bath arranged downstream of the encapsulation device to collect the cellular microcompartments formed by the encapsulation device and arranged to cause stiffening of the outer layer of each cellular microcompartment during its immersion in the bath.

[0011] The encapsulation system is characterized in that it comprises at least one member capable of electrically charging at least one of the solutions with a first electrical potential and in that the stiffening bath is electrically charged with a second predetermined electrical potential.

[0012] It is thus understood that the invention proposes to improve the cell encapsulation system according to the prior art, by electrically charging the stiffening bath with a predetermined electrical potential, in particular distinct from that with which the microcompartments are charged. Thanks to this characteristic, the electrical charges carried by these microcompartments flow from these microcompartments towards the source of this second electrical potential, which avoids the accumulation of charges and the associated problems and then makes it possible to increase the yield and quality of the cell culture within the microcompartments.

[0013] In an embodiment according to the invention, it may be provided that the system comprises a third container comprising an intermediate solution, such as an isotonic intermediate solution, preferably an isotonic solution not containing a divalent cation such as Ca2+ Mg2+ to avoid crosslinking of the hydrogel too early in the injector, such as for example a sorbitol solution.

[0014] According to one variant, the cell solution may contain culture medium and / or an extracellular matrix and / or an extracellular matrix substitute and / or an aqueous solution. According to another variant, the intermediate solution may contain an extracellular matrix and / or an extracellular matrix substitute. Where appropriate, the device will be arranged to form, at the outlet of the encapsulation device, from the solutions supplied by the dispenser(s), cellular microcompartments whose outer layer is the solution capable of gelling, an intermediate layer forming a cellular matrix or extracellular matrix substitute and the core the cell solution. This cellular matrix allows the cells of the cell solution to develop and multiply.For example, the extracellular matrix substitute may comprise a mixture of proteins and extracellular compounds necessary for cell culture, and more particularly for pluripotent cells. Preferably, the extracellular matrix or extracellular matrix substitute may comprise structural proteins, such as laminins containing the α1, α4 or α5 subunits, the β3L or β32 subunits, and the γ1 or γ3 subunits, entactin, vitronectin, laminins, collagen, as well as growth factors, such as TGF-beta and / or EGF. The extracellular matrix may be an aqueous solution and / or a hydrogel, preferably a hydrogel, different from the hydrogel forming the outer layer, such as for example a hydrogel comprising or consisting of alginate, fibrin, laminin, fibronectin, entactin, hyaluronic acid and / or collagen.It may also be an extracellular matrix or an extracellular matrix substitute such as Matrigel®. In the invention, the cell solution comprises a plurality of cells. The cells may be of any cell type. More preferably, the cells are chosen from human, animal and plant eukaryotic cells, even more preferably pluripotent stem cells, progenitors, cells undergoing differentiation and differentiated cells. Where appropriate, said pluripotent stem cells may be induced pluripotent stem (IPS) cells, MUSE (Multilineage-differentiating Stress Enduring) cells found in the skin and bone marrow of adult mammals, or embryonic stem (ES) cells.In a particular embodiment, and for legal or ethical reasons, stem cells are understood to exclude human embryonic stem cells or cells requiring the destruction of human embryos.

[0015] In an exemplary embodiment of the invention, the gel-capable solution comprises or consists of a hydrogel, such that the outer layer is a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, and preferably water. For example, the solution capable of gelling comprises or consists of alginate and, preferably, consists of alginate. In the context of the invention, the term "alginate" means linear polysaccharides formed from |3-D-mannuronate (M) and aL-guluronate (G), salts and derivatives thereof. Advantageously, the alginate is a sodium alginate, composed of more than 80% G and less than 20% M, with an average molecular mass of 100 to 400 KDa and a total concentration of between 0.5% and 5% by mass.

[0016] In the microcompartments obtained by means of the system according to the invention, the cells present in the internal part can be isolated and / or in the form of at least one layer and / or or in the form of at least one three-dimensional aggregate and / or in the form of at least one three-dimensional cellular micro-tissue, optionally with at least one lumen.

[0017] According to a variant, at least one cellular microcompartment obtained by means of the system according to the invention comprises at least one layer of cells and at least one lumen. When the microcompartment comprises at least one lumen, at least one layer of cells, the intermediate solution layer of the internal part and the external layer are preferentially, are successively organized around said lumen. This is referred to as a cyst-shaped conformation. Thus, according to a variant, at least one cellular microcompartment obtained by means of the system according to the invention comprises at least one cyst, the hollow center, or lumen, of which is preferentially aqueous. In the context of the invention, a "cyst" means a three-dimensional arrangement in a monolayer of cells or in an epithelial layer, spherical, surrounding a central lumen. This conformation in the form of cyst(s) makes it possible to reduce the pressures experienced by the cells.This configuration also allows to reduce cell mortality and increase the amplification factor of the culture. Consequently, this allows to reduce the number of passages and dissociation necessary; to reduce the time in culture necessary to reach the final number of cells required.

[0018] The cellular microcompartments obtained by means of the system according to the invention preferably comprise one or more cysts, and / or one or more tissues and / or microtissues and / or cell aggregates with or without lumen(s).

[0019] Advantageously, the encapsulation system is arranged so that each cellular microcompartment obtained by means of this system is closed. In one embodiment, the encapsulation system is arranged so that each cellular microcompartment obtained by means of this system has a spherical or drop shape. Preferably, the diameter of such a microcompartment is between 10 μm and 1 mm, more preferably between 50 μm and 700 μm, even more preferably greater than 200 μm, preferably less than 600 μm.

[0020] In another embodiment, the encapsulation system may be arranged such that each cellular microcompartment obtained by means of this system has a elongated shape, especially an ovoid or tubular shape.

[0021] Advantageously, each container may be a syringe or a tube with a conical bottom. Advantageously, each container is connected to an inlet of the encapsulation device by one or more conduits, pipes or tubes, a syringe pump or a peristaltic pump making it possible to distribute, continuously or by dose, the solution contained in this container to said inlet of the encapsulation device.

[0022] In one embodiment of the invention, it may be provided that the electrically charged solution with said first electrical potential is the solution capable of gelling. Alternatively or cumulatively, the electrically charged solution with said first electrical potential may be an intermediate solution, intended to form a layer between the outer layer and the core. Alternatively or cumulatively, the electrically charged solution with said first electrical potential may be the cell solution. According to one example, said member capable of charging one of the solutions with the first electrical potential may comprise an electrode immersed in the container containing this solution and immersed in this solution. Alternatively, said member may be a component of the dispenser intended to convey said solution to the encapsulation device and in contact with this solution.For example, it may be provided that said solution is charged with a direct current between -10KV and +10KV, preferably between -5KV and +5KV, in particular between +1.5KV and 4kV.

[0023] In one embodiment, the encapsulation device may be a microfluidic type device capable of generating a concentric jet comprising the cell solution in the center, where appropriate surrounded by the intermediate solution, itself surrounded where appropriate by the solution capable of gelling. The increase in hydrodynamic instabilities in the jet forces the jet to fragment into drops, this effect being known as Plateau-Rayleigh instability. These drops, once immersed in the bath, form the cellular microcompartments. Charging at least one of the solutions passing through the micro-injector makes it possible to improve the breaking of the jet into drops. This technique is notably called "electro-jetting". Alternatively, it may be provided that the drops form directly (without a jet) at the outlet of the injector, this technique being notably called "electro-dripping".Whatever the embodiment envisaged, the injector outlet may be arranged above the stiffening bath, so that the microcompartments fall by gravity into this stiffening bath.

[0024] In the case of electro-jetting, it may be possible to add an electric field generating device, such as a metal ring placed downstream of the outlet of the encapsulation device so that the jet or the cellular microcompartments pass through this ring. If necessary, the electric field generating device may be connected to an electric potential, for example to ground. This electric field helps in particular to promote the dispersion of the cellular microcompartments.

[0025] The stiffening bath can be arranged to trigger or to participate in the gelation of the outer layer. For example, the bath may contain a calcium solution to crosslink an alginate solution. Preferably, the system may include an electrode connected to the second electrical potential and immersed in the stiffening bath.

[0026] In one embodiment of the invention, the stiffening bath is electrically charged with a second electrical potential distinct from the first electrical potential. For example, the second electrical potential may be substantially constant, and in particular lower than the first electrical potential. This characteristic makes it possible to prevent the charges carried by the drops from flowing in the bath towards the source of the second electrical potential rather than accumulating in the bath, which would have the effect of modifying the potential of the bath and therefore its capacity to collect the capsules, or even leading to a modification of the shape of the capsules or a loss of integrity of the capsules.

[0027] Advantageously, the second electrical potential may be a zero potential. If necessary, said electrode connected to the second electrical potential and immersed in the stiffening bath may be connected to ground.

[0028] Alternatively, the second electrical potential may be non-zero and opposite in sign to the first electrical potential. This characteristic allows the bath to be charged with a potential capable of attracting the capsules during their collection.

[0029] Alternatively, the second electrical potential may be non-zero and identical in sign to the first electrical potential. This characteristic makes it possible to charge the bath with a potential capable of slowing down the capsules during their collection.

[0030] In another exemplary embodiment, the second electrical potential may be identical to the first electrical potential.

[0031] In an exemplary embodiment of the invention, the stiffening bath is connected to the second electrical potential through equipment for measuring at least one characteristic of an electric current created by the movement of the electrical charges carried by a cellular microcompartment towards said second electrical potential during its immersion in the bath. Said measuring equipment may for example be arranged to measure the intensity of an electric current created by the movement of these electrical charges, to measure a potential difference created by the movement of these electrical charges, or even to measure a quantity of electrical charges moving towards the second electrical potential.

[0032] In this example, the measuring equipment makes it possible to carry out a control of the performance and / or the quality of the encapsulation process implemented by the system according to the invention. Indeed, it has been established that the electric current created by the movement of the electric charges carried by a cellular microcompartment during its immersion in the bath is directly linked to the electric charges carried by the cellular microcompartment, which themselves depend on multiple factors including the size of the microcompartment, the proportions of solutions that compose it, the composition of each of these solutions and the flow rate of the solutions during the implementation of the encapsulation process. Therefore, the measurement of this current, or its variations, makes it possible to monitor the quantity of microcompartments produced by the encapsulation device, and / or the quality of the microcompartments, as they are produced, and / or the quality and efficiency of the implementation of the encapsulation process without using expensive equipment, such as a high-frequency camera which would film the production of the microcompartments.

[0033] Advantageously, the system comprises a calculation unit arranged to compare the measurement made by the measuring equipment with a set value and, in the event of a drift of said measurement with respect to said set value, to issue an alert indicating a fault during the implementation of an encapsulation method by the encapsulation system. It may be provided that the calculation unit issues an alert when said measurement exceeds a threshold value, high or low, or when the variation of said measurement, between two successive acquisitions, exceeds a threshold value, high or low, or when said measurement, alone or combined with one or more parameters of the encapsulation system, approaches or deviates from a predetermined law.The calculation unit will thus be able to detect the appearance of a defect, such as a modification of a flow rate of one of the solutions upstream of the encapsulation device, a change in the concentration of a compound of one of the solutions upstream of the encapsulation device, a variation in the flow rate of a jet formed by the encapsulation device, wetting of a tip of the encapsulation device, the appearance of a bubble in a distribution line connecting a container to the encapsulation device, or a defect in the stiffening bath.

[0034] Alternatively or cumulatively, the calculation unit may be arranged to detect and identify a production event from the evolution of the measurement carried out by the measuring equipment. The calculation unit may thus detect in particular the electrical charging of the solution with the first electrical potential of the solution upstream of a production cycle, the collection of the first capsule in the stiffening bath at the start of a production cycle of a new batch, the change of solution in the stiffening bath, the advent of a stable regime of capsule production, a reduction of the first electrical potential to initiate the end of a production cycle.

[0035] For example, said alert may be a visual alert displayed instantly on a control terminal of the system according to the invention, this terminal being able to be remote or not from the encapsulation device. Alternatively, the alert may be stored in a monitoring log of the production of a batch of cellular microcompartments using the system according to the invention.

[0036] Advantageously, the system comprises at least one device for measuring a value of a parameter of the encapsulation process, in that the calculation unit is arranged to determine a type of fault from the measurements made by the measuring device and the measuring equipment, said alert emitted by the calculation unit containing said type of fault. For example, the measuring device may be arranged to measure the flow rate of one or of several of the solutions in the dispensers. Alternatively, the measuring device may be arranged to acquire images of the microcompartments formed by the encapsulation device, and to determine at least one dimension of said microcompartments from said acquired images. Where appropriate, the measuring device may comprise a camera arranged at a tip of the encapsulation device.

[0037] The joint use of this device for measuring a value of a parameter of the encapsulation process and the equipment for measuring at least one characteristic of an electric current created by the movement of the electric charges carried by a cellular microcompartment during its immersion in the bath makes it possible to easily discriminate a defect in the implementation of the process by the system according to the invention. For example, there is a correlation between the flow rate of the solutions and said electric current. Therefore, it is possible to conclude, in the presence of a measured flow rate in accordance with a setpoint and an electric current whose value varies with respect to an expected value, that the tip of the encapsulation device is subject to a wetting effect.

[0038] In one embodiment of the invention, the or each dispenser comprises a device for controlling the flow rate of solution supplied by this dispenser. Where appropriate, the system according to the invention comprises a control unit for the control device(s), the control unit being capable of receiving said alert indicating a fault and being arranged, upon receipt of said alert, to control the or at least one of said control devices so as to modify or stop the flow rate of solutions by the or at least one of the dispensers. In other words, the system according to the invention benefits from a control loop and is thus capable of regulating one of the parameters likely to introduce a production fault. For example, it may be provided that the system is capable of slowing down the flow rate of dispensers, or even completely stopping distribution in order to stop the process and resolve the detected fault.

[0039] In one embodiment of the invention, the system comprises a calculation unit arranged to estimate, from the measurements made by the measuring equipment during a given period of time, a value of a geometric or quantitative parameter of the cellular microcompartments collected by the stiffening bath during said period of time.

[0040] In an exemplary embodiment, the calculation unit may be arranged to count and / or control the number of cellular microcompartments collected by the stiffening bath during said period of time. Indeed, in the case where the measurement carried out by the measuring equipment is sufficiently temporally resolved, the collection of each cellular microcompartment in the stiffening bath can be observed through the circulation of charge created by this collection. It is also possible to measure the number of electrical charges per microcompartment and to qualify the dispersion or homogeneity of the collected microcompartments, as well as to identify the occurrence of events likely to alter this number of electrical charges per microcompartment. Conversely, if the measurement resolution is not sufficient to be able to discreetly observe the collection of micro-compartments, it will still be possible to observe production events and / or control the production process by observing the evolution of the measured current.

[0041] For example, it has been found that the production of microcompartments by the encapsulation device is constant over time, particularly in a so-called "electro-dripping" regime. Therefore, the accumulation of charges in the bath is normally linear over time, and the measurements made by the measuring equipment during a given period of time therefore evolve in steps, which makes it possible to count the number of cellular microcompartments collected by the stiffening bath during this period of time.The calculation unit can thus, beyond this number of microcompartments collected, derive certain information relating to the performance and quality of the encapsulation process implemented by the encapsulation system, such as a percentage of cellular microcompartments collected by the stiffening bath compared to a number of cellular microcompartments produced by the encapsulation device, either calculated theoretically from the flow rates of the solutions and parameters relating to the structure of the encapsulation device, or measured from other measuring equipment, such as a camera.

[0042] The computing unit may also determine information relating to the homogeneity of the production of the microcompartments and the size of the microcompartments. In another example, the geometric parameter may be an average diameter or the evolution of an average diameter of the cellular microcompartments formed by the encapsulation device during a production period of a batch. In another example, the quantitative parameter may be an average electrical charge or the evolution of the electrical charge carried by each cellular microcompartment formed by the encapsulation device during a production period of a batch.In another example, the quantitative parameter may be the number of cellular microcompartments formed by the encapsulation device during a production time of a batch, an average duration or the evolution of an average duration of formation of a cellular microcompartment by the encapsulation device during a production time of a batch, or even the dispersion of the average diameter of the cellular microcompartments formed by the encapsulation device, around an average value, during a production time of a batch.

[0043] Advantageously, the calculation unit is arranged to estimate, from the measurements made by the measuring equipment during said given period of time, an average dimension of the cellular microcompartments collected by the stiffening bath during said period of time. For example, said average dimension may be an average diameter of the cellular microcompartments. It will be noted that the diameter of a spherical microcompartment may for example be obtained by means of the following equation:

[0044] [Math. 1] d = where d is the diameter of the microcompartment, A is a constant depending of the system sizing, Q the total flow rate setpoint of the solutions, V the first electrical potential and i the measurement of the electrical current in the stiffening bath.

[0045] In one embodiment of the invention, the measuring equipment comprises a voltmeter connected, on the one hand, to said electrical ground and, on the other hand, to an electrode immersed in the stiffening bath, the voltmeter being connected to said electrode via a voltage follower assembly. In this example, the second electrical potential is thus the electrical ground. Advantageously, the voltage follower assembly comprises a measuring resistor of at least 1 Mohm. This assembly thus makes it possible to measure low currents, of the order of nA. Alternatively, it may be provided that the measuring equipment comprises an ammeter whose accuracy is of the order of nA.

[0046] In one embodiment of the invention, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the distributor(s), an external flow of which is the solution capable of gelling and an internal flow of which is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, the encapsulation device being arranged to form, at the outlet of the nozzle, a concentric jet from the concentric flow so that this jet splits into cellular microcompartments. The encapsulation device is thus of the “electro-jetting” type, the jet forming itself splits, by Plateau-Rayleigh instability, into microcompartments. It will be noted that the relative sizes of the outer layer and the core of the microcompartments can be adjusted by modifying the flow rate ratios of the two solutions at the distributors.

[0047] Alternatively, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), an external flow of which is the solution capable of gelling and an internal flow of which is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, the encapsulation device being arranged to form, directly at the outlet of the nozzle, said cellular microcompartments from the concentric flow. The encapsulation device is thus of the “electro-dripping” type, and thus forms the microcompartments one after the other directly from the nozzle. It will be noted that the fact that the bath is charged with a second electrical potential makes it possible to attract each microcompartment thus formed, and that the first and second electrical potentials make it possible to control the size of the microcompartments.

[0048] Regardless of the embodiment considered, it may be provided that the body and / or the nozzle are made of glass. Alternatively, the body and / or the nozzle may be made of polymer or metal. It may be provided that the body and the nozzle form a single piece or that the body and the nozzle are made separately and then assembled to form the encapsulation device.

[0049] Advantageously, the body comprises a first inlet connected to a first distributor to receive the cell solution and at least one second inlet connected to a second distributor to receive the solution capable of gelling, as well as a single outlet connected to the nozzle, the body comprising a main channel comprising a substantially rectilinear portion defining a central axis of the encapsulation device, the main channel connecting the first inlet to the single outlet and at least one secondary channel connecting the second inlet to the single outlet, said secondary channel being subdivided into portions extending around the first channel, said subdivisions of the second channel joining at the single outlet in a single circular portion, concentric with the first channel, said single circular portion and the first channel joining to form the single outlet of the body.

[0050] The invention also relates to a cell encapsulation system, the system comprising at least: a. two containers, one of the containers comprising a cell solution and the other of the containers comprising a solution capable of gelling, b. an encapsulation device provided with several inlets each connected to one of the containers via at least one distributor and arranged to form, at the outlet of the encapsulation device, from the solutions supplied by the distributor(s), cellular microcompartments whose outer layer is the solution capable of gelling and the core the cell solution, c. a stiffening bath arranged downstream of the encapsulation device to collect the cellular microcompartments formed by the encapsulation device and arranged to cause stiffening of the outer layer of each cellular microcompartment during its immersion in the bath.

[0051] The encapsulation system is characterized in that it comprises at least one member capable of electrically charging at least one of the solutions with a first electrical potential and equipment for measuring at least one characteristic of an electric current created by the movement of the electrical charges carried by a cellular microcompartment in the bath during immersion of the microcompartment in the bath.

[0052] The present invention is now described using examples which are purely illustrative and in no way limitative of the scope of the invention, and from the appended drawings, drawings in which the various figures represent:

[0053] [Fig. 1] represents, schematically and partially, a view of an encapsulation system according to an embodiment of the invention;

[0054] [Fig. 2] represents, schematically and partially, an example of the realization of measuring equipment of the system of [Fig. 1];

[0055] [Fig. 3] represents, schematically and partially, a curve of the evolution of measurements carried out using the measuring equipment of [Fig. 2];

[0056] [Fig. 4] shows, schematically and partially, other measurements carried out using the measuring equipment of [Fig. 2];

[0057] [Fig. 5] represents, schematically and partially, other measurements carried out using the measuring equipment of [Fig. 2];

[0058] [Fig. 6] shows, schematically and partially, other measurements made using the measuring equipment of [Fig. 2]; and

[0059] [Fig. 7] represents, schematically and partially, a view of an encapsulation system according to another embodiment of the invention.

[0060] In the following description, elements which are identical, by structure or by function, appearing in different figures retain, unless otherwise specified, the same references.

[0061] [Fig. 1] shows a cell encapsulation system 1 according to a first embodiment of the invention.

[0062] The system 1 comprises two containers 21 and 22. A first container 21 comprises a solution S1 comprising a plurality of human pluripotent stem cells. A second container 22 comprises a solution S2 capable of gelling, the solution S2 comprising for example a hydrogel such as alginate.

[0063] The system 1 also comprises an encapsulation device 3 arranged to form, from the solutions SI and S2 supplied by the distributor(s), cellular microcompartments MC whose outer layer is the alginate solution S2 and the core the cell solution SI.

[0064] For these purposes, the encapsulation device 3 comprises several inlets 31, 32 each connected to one of the containers 21, 22 via a distributor 21a, 22a. Each distributor 21a, 22a thus comprises a distribution circuit and a displacement member, such as a syringe pump or a peristaltic pump, making it possible to distribute, continuously or by dose, the solution S1, S2 contained in a container 21, 22 to the inlet 31, 32 to which it is connected. In the example described, each distributor 21a, 22a is provided with a member for controlling the displacement member, making it possible to control the flow rate of the solution S1, S2 supplied by this distributor to the encapsulation device 3.

[0065] It should be noted that the dispenser 22a intended for dispensing the alginate solution S2 is equipped, in the example described, with a member 22b capable of electrically charging the alginate solution S2 with a first electrical potential VI. It may be provided, as a variant, that the alginate solution S2 is charged directly into its container 22, via an electrode immersed in this solution S2.

[0066] The encapsulation device 3 is a microfluidic device which comprises a body 33, comprising the inlets 31, 32, and a nozzle 34 connected to a single outlet of the body 33 and forming a single outlet of the device 3. It may be provided that the body 33 and the nozzle 34 are made of glass or another material suitable for the pharmaceutical industry. It may be provided that the body 33 and the nozzle 34 form a single piece or, on the contrary, that they are made separately and then assembled to form the encapsulation device 3.

[0067] In the example described, the body 33 comprising a main channel comprising a portion substantially rectilinear defining a central axis of the encapsulation device 3. This main channel connects the first inlet 31 to the single outlet of the body 33. The body 33 comprises a secondary channel connecting the second inlet 32 ​​to the single outlet. This secondary channel is subdivided into portions extending around the first channel, said subdivisions of the second channel joining at the single outlet of the body 33 in a single circular portion, concentric with the first channel. This single circular portion and the first channel then join to form the single outlet of the body 33.

[0068] In other words, the body 33 makes it possible to form a concentric flow from the solutions SI and S2 supplied by the distributor(s) 21a, 22a, an external flow being formed by the alginate solution S2 and an internal flow being formed by the cell solution SI.

[0069] The nozzle 34 thus receives the concentric flow. Taking into account the flow rates of the solutions SI and S2, and the electrostatic force generated by the electrical charges carried by the solution S2, the encapsulation device 3 thus generates, at the outlet of the nozzle, a concentric jet JT from the concentric flow. This concentric jet JT is split, under the effect of the Plateau-Rayleigh instability, into cellular microcompartments MC whose outer layer is the alginate solution S2 and the core the cell solution SI.

[0070] The encapsulation device 3 is thus of the “electro-jetting” type. It will be noted that the relative sizes of the outer layer and the core of the microcompartments MC can be adjusted by modifying the flow rate ratios of the two solutions SI, S2 using the control members of the distributors 21a, 22a, while the overall size of the microcompartments MC can be controlled by adjusting the overall flow rate of the solutions SI, S2 and the first electrical potential VI.

[0071] Alternatively, it may be possible to size the flow rates of the solutions SI and S2 as well as the first electrical potential so that the encapsulation device 3 is of the “electro-dripping” type, and thus forms the microcompartments MC one after the other directly from the nozzle 34.

[0072] In the case of an "electro-jetting" type device, it will be possible to add a metal ring connected to ground downstream of the outlet of the encapsulation device so that the JT jet or the MC cellular microcompartments pass through this ring. The electric field generated by this metal ring may help to promote the dispersion of the MC cellular microcompartments, in the case of an "electro-jetting" type device.

[0073] The system 1 comprises a stiffening bath 4 arranged under the encapsulation device 3 to collect the cellular microcompartments MC formed by this encapsulation device 3 and falling by gravity.

[0074] This stiffening bath 4 comprises a calcium solution 41 making it possible to crosslink an alginate solution S2, and thus making it possible to cause stiffening of the outer layer of each cellular microcompartment MC during its immersion in the bath.

[0075] Each cellular microcompartment MC obtained by means of this system 1 is thus closed and has, in the example described, a spherical or elongated drop shape.

[0076] Alternatively, it may be provided that the system 1 comprises a third container comprising an intermediate solution, for example comprising an isotonic solution such as sorbitol, and connected to a third inlet of the encapsulation device 3.

[0077] In a variant, the container comprising the cell solution and / or the container comprising the intermediate solution, comprises at least one extracellular matrix and / or an extracellular matrix substitute. In this case, the encapsulation device may form cellular microcompartments MC including the outer hydrogel layer originating from the solution S2, an intermediate layer forming a cellular matrix and / or an extracellular matrix substitute originating from the solution SI and / or the intermediate solution and at least one layer and / or layer and / or aggregate and / or cyst of cells originating from the cell solution SI. In this example, it may be envisaged that the solution electrically charged with said first electrical potential VI is the alginate solution S2 and / or the intermediate sorbitol solution.

[0078] In the described embodiment, the calcium solution 41 is electrically charged with a second electrical potential V2 lower than the first electrical potential VI. For this purpose, an electrode 51 is connected to the second electrical potential V2 and immersed in the calcium solution 41. In the described example, the second electrical potential V2 is a zero potential and the electrode 51 is connected to ground.

[0079] The electrical charges carried by the MC microcompartments thus flow, when immersed in the calcium 41 solution, towards this second electrical potential V2, which neutralizes the MC microcompartments. Thus, in the presence of electrical charge, the risk of these MC microcompartments merging is reduced.

[0080] In the example described, the electrode 51 is connected to the second electrical potential V2 through measuring equipment 52. This equipment 52 makes it possible to measure a characteristic of an electrical current created by the displacement of the electrical charges carried by the alginate solution S2 from a cellular microcompartment MC to said second electrical potential V2, when this cellular microcompartment MC is immersed in the calcium solution 41.

[0081] [Fig. 2] shows an exemplary embodiment of the measuring equipment 52. In this example, the measuring equipment 52 comprises a voltmeter 61 connected, on the one hand, to the electrical potential V2 and, on the other hand, to the electrode 51 via a voltage follower system 62, comprising in particular an operational amplifier. The electrode 51 is also connected to the electrical ground via a second resistor 63. The internal resistance of the system 62 is chosen to be substantially greater than the resistor 63, so that the electrical charges flowing from the bath preferentially pass through the resistor 63, which thus forms a measuring resistor. The voltmeter 61 can thus estimate the voltage between the terminals of this resistor 63 and therefore estimate the electrical current 11 which flows through it, this current being that generated by the flow of the charges carried by the cellular microcompartments MC towards the second electrical potential V2. Taking into account the order of magnitude of this current, the value of the measuring resistor 63 is at least 1 Mohm.

[0082] Alternatively, it may be provided that the measuring equipment 52 comprises an ammeter whose precision is of the order of nA, in order to directly measure the current 11. However, it has been established that the precision of the equipment 52 shown in [Fig. 2] is substantially equivalent to that of such an ammeter.

[0083] Alternatively, it may be provided that the measuring equipment 52 makes it possible to measure other characteristics, such as the quantity of electrical charges moving towards the second electrical potential V2.

[0084] In the example of [Fig. 1], the system 1 comprises a calculation unit 71 arranged to identify the appearance of particular variations, peaks and / or drops in the measurement carried out by the measurement team 52 in order to detect given events during a production cycle.

[0085] [Fig. 3] shows an example of the evolution of the intensity of current 11 measured along a production cycle of microcompartments MC.As shown on this curve, the calculation unit 71 can for example detect an initial peak in the current 11, corresponding to a first event (i), namely the electrical charging of the alginate solution S2 with the first electrical potential VI to initiate the production cycle of the batch, then a significant variation in the current 11 in a stable regime, corresponding to a second event (ii), namely the appearance of a bubble in the distributor 22a, then a reduction in the current 11 below a first threshold value, corresponding to a third event (iii), namely a reduction in the first electrical potential VI to initiate the end of the production cycle of the batch, and a reduction in the current below a second threshold value, corresponding to a fourth event (iv), namely the end of the production cycle.

[0086] The calculation unit 71 may also be arranged to compare the measurement carried out by the measuring equipment 52 with a set value.

[0087] For example, this could be a direct comparison of the value of current 11 to an expected value.

[0088] Alternatively, the calculation unit 71 may estimate a value of the flow rate of the solutions SI and S2 from the estimated value of the current 11. As shown in [Fig. 4], it has in fact established a direct linear relationship between the value of the current 11 and the value of the flow rate of the solutions SI and S2. Based on this linear relationship, the calculation unit 71 can thus estimate a value of the flow rate of the solutions SI and S2 and compare it to the setpoint of the distributors 21a and 22a. In the event of a drift, the calculation unit 71 can thus conclude that there is a fault in the encapsulation device 3, such as for example a wetting effect at the tip 34.

[0089] In this case, the calculation unit 71 may issue an alert indicating a fault during the implementation of an encapsulation method by the encapsulation system 1. For example, said alert may be a visual alert displayed instantly on a terminal. control 7 of system 1, allowing an operator to control the quality of the encapsulation process and, if necessary, to stop the execution of the process. Alternatively, the alert may be stored in a production monitoring log of a batch of cellular microcompartments using system 1, in particular for the purposes of certification or qualification of said batch.

[0090] It may also be a comparison of the value of the current 11, combined with a value of a parameter of the encapsulation process implemented by the encapsulation system 1, with given ranges of values ​​or with a given law.

[0091] [Fig. 5] shows different measurements of the current 11 as a function of the value of an electrical voltage of a metal ring (not shown in [Fig. 1]) arranged downstream of the outlet of the encapsulation device 3 so that the jet or the cellular microcompartments MC pass through this ring in order to be collected by the bath 4.

[0092] The solid line curves represent the evolution of the current 11 when the tip 34 is wetted, while the dotted line curves represent the evolution of the current 11 when the tip 34 is not wetted (the measurements having been acquired in these two cases twice, for the same encapsulation device 3). These solid and dotted line curves being clearly distinct, the calculation unit 71 can therefore segment, from the measurement of the current 11, these two scenarios of wetting and non-wetting of the tip 34.

[0093] As a further variant, the calculation unit 71 may determine an average value of the diameter of the cellular microcompartments MC formed by the encapsulation device 3 during a production period of a batch, from the measurements of the electric current 11 carried out by the measuring equipment 52 during this period. In particular, reference may be made to the equation [Math. 1] to obtain a relationship between the measurement of the electric current 11 and the diameter of a cellular microcompartment MC of spherical shape.

[0094] In the case where the characteristic measured by the measuring equipment 52 is the quantity of electrical charges moving towards the second electrical potential V2, it may be envisaged that the calculation unit 71 determines, from this quantity, the number of cellular microcompartments MC formed by the encapsulation device 3 during a production time of a batch, an average duration or the evolution of an average duration of formation of a cellular microcompartment MC by the encapsulation device 3 during a production time of a batch, or even the dispersion of the average diameter of the cellular microcompartments MC formed by the encapsulation device 3, around an average value, during a production time of a batch.

[0095] [Fig. 6] shows an example of the evolution of the accumulation of electrical charges in the calcium solution 41, measured along a production cycle of microcompartments MC for example using the derivative of the current 11. This evolution is thus represented in the form of a staircase curve, each step corresponding to the collection of a microcompartment MC by the bath 4. The calculation unit 71 can thus count, from the number of steps, the number of microcompartments collected. The calculation unit 71 will also be able to determine a ratio between this number of microcompartments collected and the number of microcompartments produced by the encapsulation device, theoretical or measured using a measuring device 53 as shown in [Fig. 7],

[0096] On the other hand, the size of each step depends on the load carried by each microcompartment MC, which is in particular a function of the dimensions of the microcompartment MC, as indicated in particular by equation [Math. 1]. The control unit can therefore distribute the different loads measured on a histogram to determine a distribution of the dimensions of the microcompartments MC in the batch of microcompartments produced, or even determine statistical moments of this distribution, such as an average diameter and a standard deviation.

[0097] [Fig. 7] shows a cell encapsulation system 10 according to a second embodiment of the invention.

[0098] This system 10 is similar to the system 1 of [Fig. 1], and further comprises a measuring device 53 for measuring a value of a parameter of the encapsulation process. This measuring device 53 could for example be a flow meter arranged at one or other of the distributors 21a, 22a, or a camera arranged at the tip 34 to acquire images of the microcompartments MC formed by the encapsulation device 3, and to determine at least one dimension of said microcompartments MC from these images.

[0099] The calculation unit 71 thus receives the measurements made by the equipment 52 and by the device 53. It can thus, in the event of detection of a drift from the measurements of the electric current II made by the equipment 52, discriminate a type of fault from these different measurements and issue an alert indicating this type of fault.

[0100] The calculation unit may in particular discriminate a modification of a flow rate of one of the solutions SI, S2 upstream of the encapsulation device 3, a change in the concentration of a compound of one of the solutions SI, S2 upstream of the encapsulation device 3, a variation in the flow rate of the jet JT formed by the encapsulation device 3, a wetting of the tip 34 of the encapsulation device 3, or even a defect in the stiffening bath 4.

[0101] More precisely, in the case where the device 53 comprises a flow meter arranged at each of the distributors 21a, 22a and in the case of detection of a significant variation in the electric current 11 without a modification of the flow rate being observed by the flow meters of the device 53, the calculation unit 71 will be able to conclude that a fault has appeared at the level of the encapsulation device 3, of the stiffening bath 4 or in a line for collecting the capsules from the bath 4. Similarly, a drift beyond the normal values ​​of the electric current 11 will allow the calculation unit 71 to conclude that there is a fault in the composition of one of the solutions S1, S2.

[0102] In the example described, the system 1 comprises a control unit 73 capable of controlling the distributors 21a and 21b. The control unit 73 can thus receive the alert issued by the calculation unit 71, and depending on the type of fault, control one and / or the other of the distributors 21a and 21b so as to modify or stop the flow of solutions SI, S2.

[0103] The foregoing description clearly explains how the invention makes it possible to achieve the objectives it has set itself, namely to propose a three-dimensional cell culture system making it possible to increase the yield and quality of the culture of these cells. It is understood that the invention proposes to improve the cell encapsulation system according to the prior art, by electrically charging the stiffening bath with a given electrical potential, in particular distinct from that with which the microcompartments are charged, which makes it possible to create a flow of charges towards the source of this other potential and therefore to avoid an accumulation of charge which would repel the microcompartments from the stiffening bath. In addition, it is possible to use this characteristic to measure characteristics of this charge flow in order to identify production defects during encapsulation.

[0104] In any event, the invention cannot be limited to the embodiments specifically described in this document, and extends in particular to all equivalent means and to any technically effective combination of these means.

Claims

Claims

1. System (1) for encapsulating cells, the system comprising at least: a. two containers (21, 22), one of the containers (21) comprising a cell solution (S1) and the other of the containers (22) comprising a solution capable of gelling (S2), b. an encapsulation device (3) provided with several inlets (31, 32) each connected to one of the containers via at least one distributor (21a, 22a) and arranged to form, at the outlet of the encapsulation device, from the solutions supplied by the distributor(s), cellular microcompartments (MC) whose outer layer is the solution capable of gelling and the core the cell solution, c.a stiffening bath (4) arranged downstream of the encapsulation device to collect the cellular microcompartments formed by the encapsulation device and arranged to cause stiffening of the outer layer of each cellular microcompartment during its immersion in the bath; characterized in that it comprises at least one member (22b) capable of electrically charging at least one of the solutions with a first electrical potential (VI) and in that the stiffening bath is electrically charged with a second predetermined electrical potential (V2).

2. System (1) according to the preceding claim, characterized in that the stiffening bath (4) is connected to the second electrical potential (V2) through equipment (52) for measuring at least one characteristic of an electric current (11) created by the movement of the electrical charges carried by a cellular microcompartment (MC) towards said second electrical potential during its immersion in the bath.

3. System (1) according to the preceding claim, characterized in that it comprises a calculation unit (71) arranged to compare the measurement carried out by the measuring equipment (52) with a set value and to, in the event of drift of said measurement with respect to said set value, emit an alert indicating a fault during the implementation of an encapsulation method by the encapsulation system.

4. System (1) according to the preceding claim, characterized in that it comprises at least one measuring device (53) for a value of a parameter of the encapsulation process, in that the calculation unit (71) is arranged to determine a type of fault from the measurements carried out by the measuring device and the measuring equipment, said alert emitted by the calculation unit containing said type of fault.

5. System (1) according to one of claims 3 or 4, characterized in that the or each dispenser (21a, 22a) comprises a member for controlling the flow rate of solution (S1, S2) supplied by this dispenser, in that it comprises a control unit (73) for the control member(s), the control unit being capable of receiving said alert indicating a fault and being arranged, upon receipt of said alert, to control the or at least one of said control members so as to modify or stop the flow rate of solutions by the or at least one of the dispensers.

6. System (1) according to one of the preceding claims, characterized in that it comprises a calculation unit (71) arranged to estimate, from the measurements carried out by the measuring equipment (52) during a given period of time, a value of a geometric or quantitative parameter of the cellular microcompartments (MC) collected by the stiffening bath (4) during said period of time.

7. System (1) according to the preceding claim, characterized in that the calculation unit (71) is arranged to estimate, from the measurements carried out by the measuring equipment (52) during said given period of time, an average dimension of the cellular microcompartments (MC) collected by the stiffening bath (4) during said period of time.

8. System (1) according to one of the preceding claims, characterized in that the measuring equipment (52) comprises a voltmeter (61) connected, on the one hand, to the second electrical potential (V2) and, on the other hand, to an electrode (51) immersed in the stiffening bath (4), the voltmeter being connected to said electrode via a voltage follower assembly (62).

9. System (1) according to one of the preceding claims, characterized in that the encapsulation device (3) comprises a body (33) arranged to form a concentric flow from the solutions (SI, S2) supplied by the distributor(s) (21a, 22a), an external flow of which is the solution capable of gelling (S2) and an internal flow of which is the cell solution (SI), and a nozzle (34) 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 (JT) from the concentric flow so that this jet is split into cellular microcompartments (MC).

10. System (1) according to one of claims 1 to 8, characterized in that the encapsulation device (3) comprises a body (33) arranged to form a concentric flow from the solutions (SI, S2) supplied by the distributor(s) (21a, 22a), an external flow of which is the solution capable of gelling (S2) and an internal flow of which is the cell solution (SI), and a nozzle (34) connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, the device encapsulation being arranged to form, directly at the outlet of the nozzle, said cellular microcompartments (MC) from the concentric flow.

11. System (1) according to one of claims 9 or 10, characterized in that the body (33) comprises a first inlet (31) connected to a first distributor (21a) for receiving the cell solution (S1) and at least one second inlet (32) connected to a second distributor (22a) for receiving the solution capable of gelling (S2), as well as a single outlet connected to the nozzle (34), the body comprising a main channel comprising a substantially rectilinear portion defining a central axis of the encapsulation device, the main channel connecting the first inlet to the single outlet and at least one secondary channel connecting the second inlet to the single outlet, said secondary channel being subdivided into portions extending around the first channel, said subdivisions of the second channel joining at the single outlet in a single circular portion, concentric with the first channel,said single circular portion and the first channel joining to form the single outlet of the body.,

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