Micro-fluidic chip and cell encapsulation system

Abstract

The invention relates to a microfluidic chip (1) consisting of a body (2), a primary inlet (5) and at least one secondary inlet (6) non-coaxial to the primary inlet (5), each primary inlet (5) and secondary inlet (6) for receiving a substance, and comprising an outlet (7), the primary inlet (5) being connected to the outlet (7) via a primary channel (8), the main channel has a substantially straight portion defining a central axis Z of the microfluidic chip (1), and the secondary inlet is also connected to the outlet (7) via a secondary channel (9). According to the invention, the secondary channel (9) is divided into at least two secondary ducts (11) of the same size, which converge in a coaxial annular injection duct with the primary channel (8) in an equally distributed manner about a central axis Z.

Description

[0001] The present invention relates to the technical field of microfluidic chips and of cell encapsulation systems comprising such microfluidic chips.

[0002] A microfluidic chip is a set of channels engraved or molded in a material such as glass, silicon or a polymer such as PDMS (Polydimethylsiloxane).

[0003] In the above-mentioned field, microfluidic chips are known which comprise a plurality of inlets for receiving various substances and a single outlet through which the substances are collected. The microfluidic chip comprises channels connecting the inlets and the outlet, the cross-section of which is in the millimeter to micrometer range.

[0004] Such microfluidic chips can be manufactured by various techniques. The most commonly used manufacturing techniques comprise the following steps: molding of a "PDMS" type material by thermoforming or by thermo-injection, or also by means of chemical lithography techniques or resin printing techniques.

[0005] Most microfluidic chips use, when in use, a liquid-liquid interface between two immiscible liquids and have a two-dimensional configuration sufficient for this use. These interfaces can be used to carry out chemical reactions (for example micro-reactors), but more often to form pellets or capsules. When a liquid-liquid interface is used, microfluidic chips are only occasionally used to form a jet, most often in droplet mode. To form a stable jet in air at the outlet of the chip, a 3D configuration is required. As with all microfluidic chips, especially for their internal structure, a high precision is required to avoid instabilities.

[0006] In the production of 3D or three-dimensional microfluidic chips, the main technique relies on 3D photopolymer resin printing. Two types of equipment can thus be used: stereolithography printers marketed on a large scale, such as "Formlab" or "Envisiontech" printers, and femtosecond laser polymerization techniques. However, the first technique allows the production of objects of several centimeters in size, but with a resolution of only around fifty micrometers, and is not able to produce robust and reproducible structures below several hundred micrometers. The second technique has the advantage of a very high spatial resolution below the micrometer, but is not industrially usable for the production of parts of centimeter size. In addition, these techniques also have the drawback of the polymerizable resins which can make them unsuitable for certain fields, such as biological production, possibly due to their chemical composition or lack of certification.

[0007] One technique for producing 3D microfluidic chips is based on subtractive 3D printing of glasses of the type fused silica, quartz, sapphire or borosilicate glass ("Selective Laser Etching"). The laser thus irradiates the parts to be removed so as to form channels in the glass plate after chemical development. This technique is intrinsically limited in height due to the working distance of the optical components, and this limitation is even more pronounced when it is necessary to achieve a high level of precision. The irradiated parts are then dissolved in an acidic or basic chemical bath. This process can last several days, so that, in order to avoid too long a residence time in the bath, it is important to limit the length of the channels. In addition to the impact on productivity and process speed, too long an immersion time can cause excessive geometric variations and dimensional changes in the structures close to the inlets and outlets. Furthermore, according to this method, the cost of the part is directly proportional to the size in the central axis Z. Finally, the presence of long channels in the chip leads to greater pressure drops, which is a real drawback for use according to the application and the liquid used.

[0008] It is therefore necessary to find a method that makes it possible to make the size in the central axis Z compact.

[0009] The above method is used to manufacture 3D microfluidic chips to produce "core-shell" type structures by double emulsion, for example by the "Raydrop" technique. Such chips comprise a 3D-printed glass head mounted on two coaxial capillaries, thus allowing 3D droplet encapsulation. Since the printing capacity and cost are limited in the Z axis, only the head is 3D-printed, which adds a complex assembly step to maintain a certain precision of the coaxial capillaries. Such structures are always carried out in a liquid phase containing immiscible liquids such as oil and water. The use of two coaxial capillaries reproduces the real fluidic resistance, which can reproduce the real limitations in its application according to the flow rate and the fluids used.

[0010] Depending on the flow rate and the nature of the substances injected into the inlets of the microfluidic chip, instabilities can exist in the various fluids that pass through the channels of the microfluidic chip. Unlike the droplet mode, the production of a jet requires a greater flow rate, which makes it more sensitive to instabilities, especially in air, which is more pronounced than in other immiscible liquids such as oil.

[0011] The present invention therefore aims to overcome these various problems and proposes a simply manufactured microfluidic chip that allows the obtaining of a coaxial jet at the outlet of the chip, composed of various incoming substances. The present invention allows the size in the Z axis direction to be made compact while providing a stable coaxial jet, and without the need for coaxial injection of fluids.

[0012] The present invention thus relates to a microfluidic chip constituted by a body comprising one primary inlet and at least one secondary inlet non-coaxial to the primary inlet, each primary and secondary inlet being intended to receive a substance, and comprising an outlet, the primary inlet being connected to the outlet by a primary channel, the primary channel having a substantially straight portion defining a central axis Z of the microfluidic chip, the secondary inlet being also connected to the outlet by a secondary channel.

[0013] The present invention thus relates to a microfluidic chip constituted by a body comprising one primary inlet and at least one secondary inlet non-coaxial to the primary inlet, each primary and secondary inlet being intended to receive a substance, and comprising an outlet, the primary inlet being connected to the outlet by a primary channel, the primary channel having a substantially straight portion defining a central axis Z of the microfluidic chip, the secondary inlet being also connected to the outlet by a secondary channel, the geometry of the primary and secondary channels being three-dimensional.

[0014] According to one embodiment, the coaxial jet at the outlet of the microfluidic chip is formed in air. Other gaseous ambient media are also compatible with the invention.

[0015] According to the invention, the secondary channel is divided into at least two secondary conduits of identical size, the secondary conduits converging in an equidistributed manner around the central axis Z in a coaxial annular injection conduit with the primary channel. The use of secondary conduits having identical hydraulic resistances allows to uniformly distribute the injection substance flow into the corresponding secondary inlets, while preserving the stability of the substance in the chip and at the outlet. All the secondary inlets comprising a secondary channel divided into secondary conduits of identical size also means that the same cross-section and shape are present in the primary channel.

[0016] All the secondary conduits from the same primary inlet converge in an equidistributed manner in an annular injection conduit coaxial to the primary channel, with respect to the primary axis, and have the same coordinates on the central axis Z, to ensure a uniform injection of the fluid around the central axis.

[0017] The three-dimensional geometry of the primary and secondary channels allows to improve the stability of the coaxial jet at the outlet. Indeed, at the outlet of the microfluidic chip, the distribution of the secondary channel around the primary channel is a three-dimensional coaxial distribution. Advantageously, the three-dimensional structure of the microfluidic chip of the invention provides a stable coaxial jet in air, and in particular of a viscous fluid.

[0018] Such a three-dimensional microfluidic chip configuration means that a substantial part of the fluid passing through the primary and secondary channels has a spatial three-dimensional component, that is to say that the fluid cannot be confined within several discrete planes, in particular within several parallel discrete planes.

[0019] The secondary injection channels from the same entry merge with the primary channel at the same coordinate of the central axis Z or at different coordinates, which allows to form a fluid composed of different substances injected through different primary and secondary entries coaxially. Forming such coaxial jet at the outlet allows to obtain an isotropic fluid at the outlet along the XY directions, avoiding instabilities. The secondary injection channels from different secondary entries do not necessarily merge with the primary channel at the same coordinate of the central axis Z.

[0020] At any point of the fluid symmetric with respect to the outlet axis at the chip outlet, the properties of the fluid are symmetric with respect to the outlet axis.

[0021] At any point of the output fluid symmetric with respect to the outlet axis, the shortest path between the point and its entry into the chip is substantially equal.

[0022] According to certain embodiments of the microfluidic chip, a non-straight portion not aligned with the central axis connects the primary entry to the primary channel. This allows to arrange the entry along an axis different from the central axis Z.

[0023] According to one feature of the application, the microfluidic chip is obtained by implementing a subtractive 3D printing technique ("Selective Laser Etching") on glass. The type of glass used is fused silica, quartz, sapphire or borosilicate glass type, and preferably fused silica. According to one embodiment, the microfluidic chip is obtained by implementing a subtractive 3D printing technique on a material allowing to perform subtractive 3D printing. Such materials allow to maintain a high precision of the channel dimensions when removed, while allowing to produce parts of millimetric volumes, up to 1 mm 3 and preferably more than 10 mm 3 , 100 mm 3 even 1000 mm 3 .

[0024] According to one embodiment of the application, the body of the microfluidic chip comprises a top face comprising the primary entry and at least one secondary entry, and a bottom face opposite the top face and comprising the outlet. The primary entry is thus aligned with the primary channel and the outlet of the chip.

[0025] According to one feature of the application, the geometry of the primary and secondary channels is three-dimensional. Thus, the geometry cannot be entirely represented in less than three two-dimensional planes.

[0026] According to one feature of the invention, the geometry of the microfluidic chip is compact in the outlet axis direction. This means that the size of all channels in the outlet axis direction will not exceed twice the size of all channels in the plane perpendicular to the outlet axis. Therefore, microfluidic chips with a size of no more than 5 cm in the Z-axis direction can be produced, and according to other embodiments, the size of the microfluidic chip in the Z-axis direction can be 1 cm or even smaller.

[0027] According to one embodiment of the present invention, the outlet shaft coincides with the central shaft Z.

[0028] According to one feature of the invention, the secondary channel includes a first diaphragm at coordinate B on the central axis Z and a second diaphragm at coordinate D on the central axis Z, thereby defining four secondary conduits of equal length. Employing a secondary channel comprising four secondary conduits of identical dimensions and hydraulic resistance allows for maximum optimization of the microfluidic chip height while providing a stable coaxial jet flow at the outlet.

[0029] According to another feature of the invention, the secondary channel includes at least one straight portion leading to the center of an arcuate portion extending in a plane perpendicular to the central axis Z. Using an arcuate diaphragm to form the secondary channel allows for a tree-like structure, thus ensuring that fluid passing through the secondary channel receives an equal path. Furthermore, this symmetry relative to the central axis Z (the coaxial axis of the microfluidic chip) allows for improved fluid stability.

[0030] According to one embodiment of the present invention, the microfluidic chip includes a first inlet and a second inlet respectively connecting a first channel and a second channel. The first channel includes an arcuate portion with a diameter of D1 extending in a plane perpendicular to the central axis Z relative to the central axis Z. The second channel includes an arcuate portion with a diameter of D2 extending in a plane perpendicular to the central axis Z relative to the central axis Z, wherein the diameter D1 is smaller than the diameter D2.

[0031] Therefore, each secondary conduit at each secondary inlet extends along an arc of a different diameter. A significant advantage of this construction is that the secondary conduits do not intersect while adhering to the principle of equal paths for each secondary channel. Thus, the dimensions of each secondary conduit vary depending on the corresponding secondary channel, but for the same primary channel, each secondary conduit corresponds to the same size.

[0032] According to one feature of the invention, the first and second secondary channels each include two arcuate portions located at two different coordinates on the central axis Z, thereby each defining four secondary conduits of the same size. This separation at the same coordinates facilitates the fabrication of microfluidic chips.

[0033] According to one embodiment of the invention, the main channel comprises four main guide tubes of the same size. The use of four main guide tubes particularly allows for a stable flow rate through the main channel.

[0034] According to another feature of the invention, each main channel and secondary channel includes a circular portion leading to an outlet, and each circular portion is concentric. The advantage of this configuration is that it creates a coaxial jet flow at the microfluidic chip outlet, which is beneficial for certain microfluidic chip applications requiring concentric fluid flow.

[0035] The present invention also relates to a cell encapsulation system having at least two containers, one containing a cell solution and the other containing a gelling solution; an encapsulation device including the microfluidic chip of the present invention, wherein a main inlet and a secondary inlet are respectively connected to one of the containers via dispensers and are capable of forming a concentric jet stream using the solution provided by the dispensers, the encapsulation device being configured such that the jet stream splits into droplets at the outlet of the encapsulation device, the outer layer of the droplets being a gelling solution and the core being a cell solution; and a gelling bath disposed downstream of the encapsulation device to collect the droplets formed by the encapsulation device, and being configured such that the outer layer of each droplet gelles upon immersion in the gelling bath.

[0036] Using this microfluidic chip is advantageous for high-volume cell encapsulation systems (compared to droplet methods), particularly due to the coaxial jet flow formed at the outlet of the microfluidic chip of this invention. The microfluidic chip of this invention can also be used for droplet output.

[0037] Using the microfluidic chip of this invention allows for the formation of coaxial jets that break into droplets at the exit of the encapsulation device, thus facilitating three-dimensional culture systems. The core of these droplets contains a cell solution completely encapsulated by the solution to be gelled. These droplets are then collected in a gelation bath, allowing their outer layer to harden and form a shell.

[0038] Three-dimensional culture more closely resembles the body's natural systems and can be used for a variety of applications, especially in therapy development. For example, for stem cell culture, gelling solutions contain alginate, while gelling baths contain calcium. The resulting encapsulated cells allow for cell culture in liquid media, while the outer shell protects the cells from the mechanical stresses caused by collisions or fusion during liquid suspension culture.

[0039] According to one variant, the cell solution may contain culture medium and / or extracellular matrix and / or extracellular matrix substitutes and / or an aqueous solution. According to another variant, the intermediate solution may contain extracellular matrix and / or extracellular matrix substitutes. If necessary, the encapsulation device will be configured to form cell microcompartments in a collection tank, with an outer layer of a gelatinable solution, an intermediate layer forming the cell matrix or extracellular matrix substitute, and an inner core of the cell solution. This cell matrix allows cells in the cell solution to grow and proliferate. For example, the extracellular matrix substitute may contain a mixture of proteins and extracellular compounds required for the culture of cells, particularly pluripotent stem cells. Preferably, the extracellular matrix or extracellular matrix substitute may include structural proteins such as laminin containing α1, α4, or α5 subunits, laminin containing β1 or β2 subunits, laminin containing γ1 or γ3 subunits, intein, hyalin, laminin, collagen, and growth factors such as TGF-β and / or EGF. The extracellular matrix can be an aqueous solution and / or a hydrogel, preferably a hydrogel, distinct from the hydrogel that forms the outer layer, such as a hydrogel comprising or composed of alginate, fibroin, laminin, fibronectin, intrin, hyaluronic acid, and / or collagen. It can also be an extracellular matrix or an extracellular matrix substitute, such as Matrigel. ® In this invention, the cell solution contains a variety of cells. The cells can be of any type. More preferably, the cells are selected from eukaryotic cells of humans, animals, and plants, and even more preferably from pluripotent stem cells, progenitor cells, differentiating cells, and differentiated cells. If necessary, the pluripotent stem cells can be induced pluripotent stem cells (IPS), MUSE cells ("Multilineage Differentiating Stress Enduring") found in the skin and bone marrow of adult mammals, or embryonic stem cells (ES). In one specific embodiment, for legal or ethical reasons, the stem cells do not include human embryonic stem cells or cells that require the destruction of human embryos.

[0040] In one embodiment of the invention, the gelatable solution comprises or is composed of a hydrogel, such that the outer layer has a three-dimensional structure formed by a polymer chain matrix formed by the expansion of a liquid (preferably water). For example, the gelatable solution comprises or is composed of alginate, and preferably alginate. In the description of the invention, "alginate" refers to a linear polysaccharide formed from β-D-mannuronic acid (M) and α-L-guluronic acid (G), as well as their salts and derivatives. Advantageously, the alginate is sodium alginate, in which the content of G is greater than 60%, even greater than 80%, the content of M is less than 40%, even less than 20%, the average molecular weight is 100 to 400 kDa, and the total mass concentration is 0.5% to 5%.

[0041] In the microcompartments obtained by the system of the present invention, the cells present in the inner portion can be separated and / or in the form of at least one layer of cells and / or in the form of at least one three-dimensional aggregate and / or in the form of at least one three-dimensional micro-tissue cell, and may have at least one cavity.

[0042] According to one variation, at least one cell microcompartment obtained by the system of the present invention comprises at least one layer of cells and at least one cavity. When the microcompartment comprises at least one cavity and at least one layer of cells, preferably an intermediate solution layer and an outer layer of the inner portion arranged sequentially around the cavity, is referred to as a vesicular configuration. Thus, according to one variation, at least one cell microcompartment obtained by the system of the present invention comprises at least one vesicle, the hollow or cavity of which is preferably aqueous. In the description of the invention, a "vesicle" refers to a spherical three-dimensional structure formed by a monolayer of cells or an epithelial layer arranged around a central cavity. This vesicular configuration allows for reduced stress on the cells. This configuration also allows for reduced cell death and increased scale-up of culture. Therefore, this allows for a reduction in the number of passages and dissociations required; and a shorter culture time required to reach the desired final cell number.

[0043] Cellular microcompartments obtained by the system of the present invention preferably contain one or more vesicles, and / or one or more tissues and / or micro-tissues and / or cell aggregates, with or without cavities.

[0044] Preferably, the system of the present invention is arranged such that each cell microcompartment obtained by the system is closed. In one embodiment, the system of the present invention is arranged such that each cell microcompartment obtained by such a system is spherical or droplet-shaped. Preferably, the diameter of such microcompartments is between 10 μm and 1 mm, more preferably between 50 μm and 700 μm, or more preferably greater than 200 μm, and preferably less than 600 μm.

[0045] In another embodiment, the system of the present invention may be arranged such that each cell microcompartment obtained by such system is elongated, particularly oval or tubular.

[0046] Advantageously, each container in the system of the present invention can be a flexible bag, a syringe, or a conical tube. Also advantageously, each container is connected to the inlet of the encapsulation device via one or more dispensers, such as via a conduit, tube, tubing, syringe plunger, or peristaltic pump, which allows for continuous or dose-based dispensing of the solution contained in the container to the inlet of the encapsulation device.

[0047] Of course, the various features, variations and embodiments of the present invention can be combined with each other in various ways, as long as they are not contradictory or mutually exclusive.

[0048] Furthermore, various other features of the invention can be learned from the accompanying drawings, which illustrate non-limiting embodiments of the invention, wherein: Figure 1 This is a perspective view of an embodiment of the microfluidic chip of the present invention, in which the internal channels are visible due to the transparency of the microfluidic chip body. Figure 2 for Figure 1 Perspective views of the microfluidic chip in the image from different angles. Figure 3 To determine the different viewpoints at the cross-section passing through coordinate A along the central axis Z, Figure 1 A half-sectional view of the microfluidic chip in the image. Figure 4 To determine the different viewpoints at the cross-section passing through coordinate B along the central axis Z, Figure 1 A half-sectional view of the microfluidic chip in the image. Figure 5 To determine the different viewpoints at the cross-section passing through coordinate C on the central axis Z, Figure 1 A half-sectional view of the microfluidic chip in the image. Figure 6 To determine the different viewpoints at the cross-section passing through coordinate D along the central axis Z, Figure 1 A half-sectional view of the microfluidic chip in the image. Figure 7 To determine the different viewpoints at the cross-section passing through coordinate E of the central axis Z, Figure 1 A half-sectional view of the microfluidic chip in the image. Figure 8 To determine the coordinates at point F along the central axis Z from different perspectives, Figure 1 A half-sectional view of the microfluidic chip in the image. Figure 9 To determine the coordinates at point G along the central axis Z from different perspectives, Figure 1 A half-sectional view of the microfluidic chip in the image. Figure 10 This is a view of an embodiment of the cell encapsulation system of the present invention. Figure 11 for Figure 10 A cross-sectional view of the encapsulation device in the image.

[0049] Figure 1 The dashed line in the diagram corresponds to the central axis Z of the microfluidic chip.

[0050] Figures 3 to 9 The thick lines in the text indicate that, Figure 1 The cross-sections marked at each coordinate from A to G on the central axis Z.

[0051] It should be noted that in these diagrams, common structural and / or functional elements of various variants may have the same identifier.

[0052] The present invention aims to stabilize the jet flow at the outlet of a microfluidic chip.

[0053] Therefore, the microfluidic chip of this invention, generally designated 1, consists of a body 2, including a top surface 3 and an opposite bottom surface 4. The top surface 3 includes a main inlet 5 and at least one secondary inlet 6. The bottom surface 4 includes an outlet 7.

[0054] The accompanying drawings illustrate a microfluidic chip 1, comprising a cylindrical body 2 about a central axis Z, having a top surface 3, a bottom surface 4, and side surfaces. Other embodiments, such as polygonal shapes, are also compatible with the invention. Therefore, the surfaces of the body 2 of the microfluidic chip 1 do not necessarily all face each other.

[0055] The main passageway 8 connects the main inlet 5 to the outlet 7. The main passageway 8 is essentially straight. The main inlet 5 is aligned with the outlet 7 along the central axis Z, as shown below. Figure 1 As shown. According to the illustrated embodiment, the main channel 8 is approximately along the central axis Z of the microfluidic chip 1.

[0056] The microfluidic chip 1 of the present invention further includes a secondary channel 9, which connects the secondary inlet 6 to the outlet 7. The microfluidic chip 1 of the present invention may include a plurality of secondary inlets 6, and the microfluidic chip 1 includes an equal number of secondary channels 9, which respectively connect each secondary inlet 6 to the outlet 7.

[0057] According to the embodiment shown, the main channel 8 and one or more secondary channels 9 include partially circular cross sections of varying sizes, as well as more complex cross sections, such as at the diaphragm that divides the channel into two parts (referred to as conduits) as described below.

[0058] Figure 1 and Figure 2 One embodiment of a microfluidic chip 1 is shown, comprising a first inlet 6a and a second inlet 6b disposed on either side of a main inlet 5, wherein the three main inlets 5 and the secondary inlets 6a, 6b are aligned. According to other embodiments of the invention (not shown), the microfluidic chip 1 includes a single secondary inlet 6, or three or more secondary inlets 6. The construction principle of the secondary channels 9 connected around the main channel 8 is the same and will be described below. According to other embodiments (not shown), the secondary inlets 6 may be misaligned.

[0059] Figure 3 Corresponding to Figure 1 In China Figure 1The cross-section is located at coordinate A on the central axis Z. The main inlet 5 is connected to the main guide tube 8. The first inlet 6a is connected to the first guide tube 9a, and the second inlet 6b is connected to the second guide tube 9b.

[0060] Figure 4 Corresponding to Figure 1 In China Figure 1 The cross-section is located at coordinate B on the central axis Z. The main channel 8 is divided into four straight and parallel main conduits 10. Furthermore, the secondary channels 9a and 9b are each divided into two secondary conduits 11a and 11b. The first channel 9a includes a straight portion close to the main channel 8, and then includes a diaphragm forming an arc, having a diameter D1 relative to the central axis Z. The second channel 9b includes a diaphragm forming an arc, having a diameter D2 relative to the central axis Z. Therefore, D1 is smaller than D2 to optimize the space contained within the plane, especially at coordinate B.

[0061] According to an embodiment not shown, the microfluidic chip 1 includes a third inlet 6 and a third channel 9. The third channel 9 includes a diaphragm forming an arc, the diameter of which, D3, is larger than the diameter D2 of the arc formed by the second channel 9. Depending on the arrangement of the third inlet, the third channel 9 includes a straight portion that allows for the arrangement of the arc, the arc corresponding to the desired diameter D3. This configuration including concentric arcs is based on the number of secondary inlets 6.

[0062] Advantageously, according to the embodiment shown, the diaphragm of each subchannel 9 is formed at the middle of the arc so as to form a subconduct 11 of the same length.

[0063] The secondary channel 9 of the microfluidic chip 1 of the present invention therefore includes at least one straight channel portion that is substantially parallel to the central axis Z.

[0064] Four catheters 11a and 11b are also present. Figure 5 As can be seen in the figure, this figure corresponds to Figure 1 In China Figure 1 The cross section marked C on the central axis Z.

[0065] According to the illustrated embodiment, the secondary channels 6a and 6b include a second separation, particularly as shown in... Figure 6 It can be seen that this corresponds to Figure 1 In China Figure 1 The cross-section is marked with coordinate D on the central axis Z. Therefore, the secondary channels 6a and 6b are divided into four secondary conduits 11a and 11b twice. This second diaphragm also includes an arcuate portion around the central axis Z of the microfluidic chip 1, with diameters D1 and D2 the same as the first diaphragm. The arcuate portions of the first and second secondary channels 9a and 9b are symmetrical.

[0066] Therefore, the four secondary ducts 11a, 11b formed in each secondary channel 9a, 9b are... Figure 7 As can be seen in the figure, this figure corresponds to Figure 1 In China Figure 1 The cross-section is located at coordinate E on the central axis Z. Therefore, twelve main channels 10 and secondary channels 11a and 11b are visible.

[0067] All main channels 8 and secondary channels 9 converge at the outlet 7 of the coaxial circular injection conduit. According to the illustrated embodiment, particularly... Figure 8 In the figure, the corresponding figure is Figure 1 In China Figure 1 A cross-section at coordinate F on the central axis Z shows each main tube 10 and secondary tubes 11a, 11b converging on their respective unique annular conduits. Each main tube 10 and secondary tubes 11a, 11b is concentric so that all tubes converge concentrically in the direction toward outlet 7 within a coaxial annular injection conduit. Therefore, all jet streams are coaxial at the outlet of the microfluidic chip 1. According to the illustrated embodiment, outlet 7 is directly exposed to air. According to other embodiments not shown, outlet 7 is connected to an auxiliary device or extension device, such as a nozzle.

[0068] According to one usage method, the microfluidic chip 1 is used in a cell encapsulation system.

[0069] The cell encapsulation system includes at least two containers, one containing a cell solution and the other containing a gelling solution.

[0070] The cell encapsulation system also includes an encapsulation device comprising the microfluidic chip 1 of the present invention, wherein a main inlet 5 and a secondary inlet 6 are respectively connected to one of the containers via a dispenser. The microfluidic chip 1 is capable of forming a concentric jet stream at the outlet using a solution provided by the dispenser. The encapsulation device is configured such that the jet stream splits into droplets at the outlet of the encapsulation device, the outer layer of the droplets being a gelatinizable solution and the core being a cell solution.

[0071] Finally, the cell encapsulation system includes a gelation bath located downstream of the encapsulation device to collect droplets formed by the encapsulation device, and is configured such that the outer layer of each droplet gels upon immersion in the gelation bath.

[0072] Figure 10 A cell encapsulation system according to an embodiment of the present invention is shown.

[0073] The system has two containers, 110 and 120. The first container 110 contains a solution containing various human pluripotent stem cells. The second container 120 contains a gelling solution, such as a hydrogel like alginate. The system may be designed to have a third container containing an intermediate solution, such as an isotonic solution like sorbitol.

[0074] The system also has an encapsulation device 130, which includes a microfluidic chip 1 and is configured to use solutions in containers 110 and 120 to form cellular microcompartments with an outer layer of alginate solution and an inner core of cell solution.

[0075] Figure 11 A cross-sectional view of the encapsulation device 130 is shown.

[0076] like Figure 11 As shown, the encapsulation device 130 has multiple inlets 5, 6, each inlet connected to one of the containers 110, 120 via a dispenser 140. In the described example, each dispenser 140 thus has a dispensing loop and a moving component, such as a pusher or peristaltic pump, which allows the dispenser to continuously or dosswise dispense the solution contained in the containers 110, 120 to the inlets 5, 6 of the device 130 to which it is connected.

[0077] It should be noted that in the described example, the dispenser 140 for dispensing the alginate solution is equipped with a device capable of charging the alginate solution using a potential. As a variation, the alginate solution can be designed to be directly charged in its container 120 via an electrode immersed in the solution.

[0078] The encapsulation device 130 includes a microfluidic chip 1. According to the illustrated embodiment, the microfluidic chip 1 has a body 2 including inlets 5 and 6, and a nozzle 340 connecting to a single outlet 7 of the body 2 and forming the single outlet of the device 130. The body 2 and nozzle 340 can be designed to be made of glass or other materials suitable for the pharmaceutical industry. Alternatively, the body 2 and nozzle 340 can be designed as a single unit, or conversely, manufactured separately and then assembled to form the encapsulation device 130.

[0079] In the described example, the body 2 has a main channel 8 having a substantially straight portion that defines the central axis of the encapsulation device 130. This main channel 8 connects a main inlet 5 to a single outlet 7 of the body 2. The body 2 has a secondary channel 9 that connects a second inlet 6 to the single outlet 7. This secondary channel 9 is further divided into portions extending around the main channel 8, and the branches of the secondary channel 9 converge at the single outlet 7 of the body 2 to form a single annular portion concentric with the main channel. This single annular portion then merges with the main channel to form the single outlet 7 of the body 2.

[0080] In other words, the microfluidic chip 1 allows the solution provided by containers 110 and 120 to form a concentric flow through dispenser 140, wherein the outflow is formed by alginate solution and the inflow is formed by cell solution.

[0081] Nozzle 340 receives the concentric flow. Taking into account the flow rate of the solution and the electrostatic force generated by the charge carried by the alginate solution, the encapsulation device 130 utilizes the concentric fluid to generate a concentric jet at the outlet of nozzle 340. This concentric jet splits into cellular microcompartments under the influence of Plateau-Rayleigh instability, with an outer layer of alginate solution and an inner core of cell solution.

[0082] Therefore, the encapsulation device 130 is of the "electro-jet" type. It should be noted that the relative size of the outer layer and the inner core of the micro-chamber can be adjusted by changing the flow ratio of the two solutions using the distributor 140, while the overall size of the micro-chamber can be controlled by adjusting the total flow rate of the solution and the potential of the alginate solution.

[0083] Another variation is to adjust the flow rate and potential of the solution so that the encapsulation device 130 becomes an "electro-dripping" type, thereby forming micro-compartments directly from the nozzle 340 one by one.

[0084] like Figure 11 As shown, a metal ring 160 grounded via connector 170 extends downstream of the outlet of encapsulation device 130, allowing the jet stream or cell microcompartments to pass through the ring. The electric field generated by the metal ring can facilitate the dispersion of cell microcompartments when using an "electro-jetting" type device.

[0085] Or refer to Figure 10 The system also includes a collection tank 150, positioned below the encapsulation device 130 and containing a first solution, for collecting cell microcompartments formed by the encapsulation device 130 and falling due to gravity. For this purpose, the collection tank 150 includes a gelling bath.

[0086] The gelling solution contains surfactants and calcium salts that allow cross-linking of the alginate solution, and thus allow each cell microcompartment to cause hardening or gelling of its outer layer when immersed in the collection tank.

[0087] Each cell microcompartment obtained through this first stage is then sealed and, in the described example, has the shape of a spherical or elongated droplet.

[0088] In one variation, the container containing the cell solution and / or the container containing the intermediate solution includes at least one extracellular matrix and / or extracellular matrix substitute. In this case, the encapsulation device 130 can form a cell microcompartment comprising an outer hydrogel layer from the alginate solution, an intermediate layer from the cell solution and / or the intermediate solution forming the cell matrix and / or extracellular matrix substitute, and at least one layer of cells and / or epithelial layer and / or cell aggregates and / or cell vesicles from the cell solution. In this example, the solution charged by the said potential may be an alginate solution and / or a sorbitol intermediate solution.

[0089] It should be noted that the collection tank 150 has a discharge port 1510 for the gelling solution and micro-chambers immersed in the solution, as well as an injection port 1520 for injecting the gelling solution into the collection tank 150. The inlet and outlet 1510, 1520 are connected to the collection circuit.

[0090] In the example, the collection tank is formed by a bottom 1530 and a top 1540. The bottom contains a gelling solution and has a discharge port 1510 and an injection port 1520. The top forms a lid on which the encapsulation device 130 and the metal ring 160 are mounted. The collection tank 1530 and the lid 1540 together constitute a closed chamber suitable for producing cell microcompartments according to specified aseptic requirements.

[0091] Of course, various other modifications can be made to the invention within the scope of the appended claims.

Claims

1. A microfluidic chip (1), the microfluidic chip comprising a body (2), the body including a main inlet (5) and at least one secondary inlet (6) non-coaxial with the main inlet (5), each of the main inlet (5) and the secondary inlet (6) for receiving material and including an outlet (7), the main inlet (5) being connected to the outlet (7) via a main channel (8), the main channel having a substantially straight portion defining a central axis Z of the microfluidic chip (1), the secondary inlets also being connected to the outlet (7) via the secondary channel (9), characterized in that, The secondary channel (9) is divided into at least two secondary conduits (11) of the same size, which merge with the main channel (8) in a distributed manner around the central axis Z in a coaxial annular injection conduit.

2. The microfluidic chip (1) according to the preceding claims is obtained by performing subtractive printing on glass.

3. The microfluidic chip (1) according to any one of the preceding claims, wherein the body (2) includes a top surface (3), the top surface including the main inlet (5) and at least one of the secondary inlets (6), and includes a bottom surface (4) opposite to the top surface (3) and including the outlet (7).

4. The microfluidic chip (1) according to any one of the preceding claims, wherein the geometry of the main channel (5) and the secondary channel (6) is three-dimensional.

5. The microfluidic chip (1) according to any one of the preceding claims, wherein the geometry of the microfluidic chip (1) is compact in the outlet axis direction.

6. The microfluidic chip (1) according to any one of the preceding claims, wherein the secondary channel (9) includes a first diaphragm at coordinate B of the central axis Z and a second diaphragm at coordinate D of the central axis Z, thereby defining four secondary conduits (11) of equal length.

7. The microfluidic chip (1) according to any one of the preceding claims, wherein the secondary channel (9) includes at least one straight portion leading to the center of an arcuate portion extending in a plane perpendicular to the central axis Z.

8. The microfluidic chip (1) according to any one of the preceding claims, the microfluidic chip comprising a first inlet and a second inlet (6a, 6b) respectively connecting a first channel and a second channel (9a, 9b), the first channel (9a) comprising an arcuate portion of diameter D1 extending relative to the central axis Z in a plane perpendicular to the central axis Z, the second channel (9b) comprising an arcuate portion of diameter D2 extending relative to the central axis Z in a plane perpendicular to the central axis Z, wherein D1 is less than D2.

9. The microfluidic chip (1) according to any one of the preceding claims, wherein the first and second secondary channels (9a, 9b) each include two arcuate portions located at two different coordinates on the central axis Z, thereby each defining four secondary conduits (11a, 11b) of the same size.

10. The microfluidic chip (1) according to any one of the preceding claims, wherein the main channel (8) comprises four main tubes (10) of the same size.

11. The microfluidic chip (1) according to any one of the preceding claims, wherein each of the main channels (8) and secondary channels (9) includes a circular portion leading to the outlet (7), and each of the circular portions is concentric.

12. A cell encapsulation system, said cell encapsulation system comprising at least: - Two containers (110, 120), one of which contains a cell solution, and the other of which contains a gelling solution. - Encapsulation device (130), the encapsulation device comprising a microfluidic chip (1) according to any one of claims 1 to 11, wherein the main inlet (5) and the secondary inlet (6) are respectively connected to one of the containers (110, 120) via a dispenser (140) and are capable of forming a concentric jet stream using the solution provided by the dispenser (140), the encapsulation device (130) being configured such that the jet stream splits into droplets at the outlet of the encapsulation device (130), the outer layer of the droplets being a gelatinable solution and the inner core being a cell solution, and - A gelling bath, located downstream of the encapsulation device (130), is provided to collect droplets formed by the encapsulation device (130) and is configured such that the outer layer of each droplet gels upon immersion in the gelling bath.

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