EP2119503B1 - Microfluid system and method for sorting clusters of cells and continuously encapsulating them once they are sorted - Google Patents

Description

The present invention relates to a microfluidic system and a method for sorting clusters of cells, such as islets of Langerhans, and for the continuous and automated encapsulation of clusters once sorted into capsules of sizes adapted to those of these sorted clusters. The invention applies in particular to the coupling between sorting and encapsulation of such clusters of cells, but also in a more general manner of cells, bacteria, organelles or liposomes, in particular.

Cell encapsulation is a technique that involves immobilizing cells or clumps of cells in microcapsules to protect them from attacks by the immune system during transplantation. The porosity of the capsules should allow entry of low molecular weight molecules essential for the metabolism of encapsulated cells, such as molecules of nutrients, oxygen, etc., while preventing the entry of higher molecular weight substances such as antibodies or cells of the immune system. This selective permeability of the capsules is thus designed to ensure the absence of direct contact between the encapsulated cells of the donor and those of the immune system of the transplant recipient, which makes it possible to limit the doses of immunosuppressive treatment used during the transplantation. treatment with severe side effects).

Among the many applications of encapsulation, we can cite that of islets of Langerhans, cluster of fragile cells located in the pancreas and consisting of several cell types, including β cells that regulate blood glucose in the body by producing insulin. The encapsulation of these islets is an alternative to conventional cell therapies (eg pancreas or islet transplantation) used to treat insulin-dependent diabetes, an autoimmune disease in which the immune system destroys its own insulin-producing β cells.

• on diminue la quantité de polymère « inutile » autour des cellules et donc le temps de réponse de ces dernières. Par exemple, la régulation de la glycémie par des îlots de Langerhans encapsulés dans des capsules de taille adaptée sera plus rapide, car le glucose diffusera plus rapidement vers l'îlot et l'insuline produite s'en échappera plus rapidement ;
• on maximise la viabilité des îlots encapsulés du fait que la diffusion de l'oxygène y est plus rapide, ce qui améliore l'oxygénation des cellules et réduit les risques d'apparition de zones nécrosées ; et
• on diminue le volume de capsules à transplanter, ce qui peut permettre l'implantation des capsules dans des zones plus propices à la revascularisation des tissus. En effet, cette revascularisation est essentielle pour éviter les nécroses des cellules encapsulées, car les cellules doivent être situées à proximité du réseau sanguin pour être bien approvisionnées en nutriments et en oxygène, notamment. Par exemple, pour le traitement du diabète insulinodépendant, ce volume réduit permet d'implanter les îlots encapsulés dans le foie ou la rate, régions plus favorables à la revascularisation que la cavité péritonéale où les capsules sont classiquement implantées pour des questions d'encombrement stérique.

The capsules produced must meet certain criteria, including biocompatibility, mechanical resistance and selective permeability, in particular. Another essential criterion is the size of the capsules, because by adjusting it better to the size of the cell clusters (see reference [1]):

• the amount of "useless" polymer around the cells is reduced and thus the response time of the latter. For example, the regulation of blood glucose by islets of Langerhans encapsulated in capsules of suitable size will be faster, because the glucose will diffuse more quickly to the islet and insulin produced will escape more quickly;
• the viability of the encapsulated islets is maximized because the diffusion of oxygen is faster there, which improves the oxygenation of the cells and reduces the risk of appearance of necrotic zones; and
• the volume of capsules to be transplanted is reduced, which may allow the implantation of the capsules in areas more conducive to revascularization of the tissues. Indeed, this revascularization is essential to avoid necrosis of encapsulated cells, because the cells must be located near the blood network to be well supplied with nutrients and oxygen, in particular. For example, for the treatment of insulin-dependent diabetes, this reduced volume makes it possible to implant the islets encapsulated in the liver or spleen, regions more favorable to revascularization than the peritoneal cavity where the capsules are conventionally implanted for steric hindrance issues. .

Although the properties of biocompatibility, mechanical resistance or selective permeability seem to be well-known from the literature, this is not the case with the size of the capsules, which is particularly problematic for the encapsulation of the islets of Langerhans. Indeed, in all the documents known to the Applicant to date, the size of the capsules formed around these islands is fixed and on average of the order of 600 to 800 microns, while these islets have a size ranging from 20 to 400 μm only. A fixed and identical size of capsules regardless of the size of the island is therefore problematic, especially since recent studies have shown that the best performing islets are the smallest (see reference [2]).

• un jet d'air ou de liquide coaxial, les capsules produites ayant une taille variant entre 400 µm et 800 µm (cependant la taille moyenne des capsules produites est plus proche de 600-800 µm que de 400 µm) ;
• une différence de potentiel, qui est la technique d'encapsulation la plus utilisée lorsque la priorité est de diminuer la taille des capsules (la taille des capsules varie dans ce cas entre 200 et 800 µm) ; ou
• une technique de vibration, qui présente l'inconvénient d'être parfois limitée par les viscosités des solutions utilisées.

The main known encapsulation methods use:

• a jet of air or coaxial liquid, the capsules produced having a size ranging between 400 microns and 800 microns (however the average size of the capsules produced is closer to 600-800 microns than 400 microns);
• a potential difference, which is the most used encapsulation technique when the priority is to reduce the size of the capsules (the size of the capsules varies in this case between 200 and 800 μm); or
• a vibration technique, which has the disadvantage of being sometimes limited by the viscosities of the solutions used.

• les tailles des capsules qui ne sont pas forcément adaptées à celles des îlots de Langerhans à encapsuler ;
• l'absence d'automatisation de la procédure d'encapsulation, où les capsules sont gélifiées en tombant dans un bain de polycations et sont ensuite récupérées manuellement, ce qui génère une hétérogénéité du temps de polymérisation d'une capsule à une autre ;
• la dispersion en taille des capsules, qui augmente lorsque la taille des gouttes diminue ; et
• un manque de reproductibilité des capsules produites, qui ne sont pas forcément sphériques.

The main disadvantages of these techniques are:

• the sizes of the capsules which are not necessarily adapted to those of the islets of Langerhans to be encapsulated;
• the lack of automation of the encapsulation procedure, where the capsules are gelled by falling into a polycation bath and are then recovered manually, which generates a heterogeneity of the polymerization time from one capsule to another;
• the capsule size dispersion, which increases as the size of the drops decreases; and
• a lack of reproducibility of the capsules produced, which are not necessarily spherical.

• ceux réalisant un tri par « Deterministic Lateral Displacement » ou « DLD » (i.e. déplacement latéral déterministe, voir références [6-8] et par exemple les documents WO-A-2004/037374 , US-A-2007/0059781 et US-A-2007/0026381 ), qui repose sur l'utilisation d'un réseau périodique d'obstacles qui vont perturber ou non la trajectoire des particules à trier. Les particules plus petites qu'une taille critique Dc, fixée par la géométrie du dispositif, ne sont globalement pas déviées par ces obstacles, tels que des plots, tandis que celles plus grandes que cette taille Dc sont déviées dans la même direction à chaque rangée de plots. La trajectoire des plus grosses particules est donc finalement déviée par rapport à celle des plus petites, ce qui permet la séparation en taille des particules, étant précisé que dans la technique « DLD » l'espacement entre deux plots adjacents est toujours supérieur à la taille des particules à dévier. Ce dispositif est adapté aux échantillons sanguins (séparation des globules rouges, blancs et du plasma) ;
• les systèmes réalisant un tri par filtration hydrodynamique (voir références [9, 10] et les documents JP-A-2007 021465 , JP-A-2006 263693 , et JP-A-2004 154747 ), qui consiste à adapter les résistances fluidiques de canaux transverses en choisissant un rapport de débits approprié entre le canal principal et ces canaux transverses. De ce fait, les particules dont la taille est supérieure à une taille critique (fixée par la valeur de la résistance fluidique) ne peuvent pénétrer dans ces canaux transverses, même si leur taille est inférieure à la largeur des canaux transverses ;
• des systèmes de tri par taille plus simples n'utilisant que la déviation des lignes d'écoulement (voir références [11, 12] et par exemple le document WO-A-2006/102258 ) où, dans la zone de tri, les lignes d'écoulement sont déviées vers une zone de basse pression: la différence de positionnement des lignes d'écoulement est accentuée, et comme les particules suivent les lignes d'écoulement sur lesquelles leur centre d'inertie est positionné, la différence de position entre petites et grosses particules est accentuée ;
• les systèmes de tri utilisant des filtres qui permettent soit de laisser passer des molécules de taille inférieure à une valeur critique (voir le document US-A-2005/0133480 ), soit de ne laisser passer que le fluide pour concentrer les particules ou séparer le fluide qui les véhicule (voir dans ce cas le document WO-A-2006/079007 ). La principale limitation de ces systèmes de tri par filtres est le risque de colmatage des canaux par les particules ; et
• les systèmes de tri où la microfluidique est couplée à un champ extérieur, comme par exemple les mesures optiques de fluorescence ou d'absorbance (voir les documents WO-A-2002/023163 et WO-A-02/40874 ), les pièges optiques, la diélectrophorèse, les mesures de conductimétrie, de potentiométrie, d'ampérométrie, les détections de liaisons ligand/récepteur, etc.

Microfluidic systems have recently been developed which are suitable for sorting by size of bacteria, cells, organelles, viruses, nucleic acids or even proteins, among which may be mentioned:

• those carrying out a sorting by "Deterministic Lateral Displacement" or "DLD" (ie deterministic lateral displacement, see references [6-8] and for example the documents WO-2004/037374 , US-2007/0059781 and US-2007/0026381 ), which relies on the use of a periodic network of obstacles that will disturb or not the trajectory of the particles to be sorted. Particles smaller than a critical size Dc, fixed by the geometry of the device, are not generally deviated by these obstacles, such as studs, while those larger than this size Dc are deflected in the same direction at each row of studs. The trajectory of the largest particles is thus finally deviated from that of the smaller ones, which allows the size separation of the particles, it being specified that in the "DLD" technique the spacing between two adjacent studs is always greater than the size particles to be deflected. This device is suitable for blood samples (separation of red, white and plasma cells);
• systems performing sorting by hydrodynamic filtration (see references [9, 10] and documents JP-A-2007 021465 , JP-A-2006 263693 , and JP-A-2004 154747 ), which consists of adapting the fluidic resistances of transverse channels by choosing a suitable flow ratio between the main channel and these transverse channels. As a result, particles whose size is greater than a critical size (fixed by the value of the fluidic resistance) can not penetrate these transverse channels, even if their size is smaller than the width of the transverse channels;
• simpler sorting systems using only the deviation of the flow lines (see references [11, 12] and for example the document WO-2006/102258 ) where, in the sorting zone, the flow lines are diverted towards a low pressure zone: the difference in the positioning of the flow lines is accentuated, and as the particles follow the flow lines on which their center of flow Inertia is positioned, the difference in position between small and large particles is accentuated;
• the sorting systems using filters that allow either to pass molecules smaller than a critical value (see document US-2005/0133480 ), either to let only the fluid to concentrate the particles or to separate the fluid which conveys them (see in this case the document WO-2006/079007 ). The main limitation of these filter sorting systems is the risk of clogging of the channels by the particles; and
• sorting systems where the microfluidic is coupled to an external field, such as optical fluorescence or absorbance measurements (see the documents WO-2002/023163 and WO-A-02/40874 ) optical traps, dielectrophoresis, conductivity measurements, potentiometry, amperometry, ligand / receptor detections, etc.

A major disadvantage of all microfluidic sorting systems presented in these documents is that they are not at all suitable for sorting clusters of cells, such as islets of Langerhans or other little cohesive clusters of similar sizes. Indeed, and as previously explained, each of these clusters behaves very differently from a cell because of its size (from 20 μm to 400 μm for islets of Langerhans against about ten μm for a single cell) and also because of its size. its weak cohesion (which imposes weak shears in the microfluidic sorting system used).

The only known system of the Applicant for sorting out such clusters of cells is the flow cytometer named "COPAS" which is marketed by Union Biometrica. This system, which is not a microfluidic type, performs size sorting of clusters by measuring their respective flight times in the beam of a laser beam (see reference [13]).

• au niveau d'une jonction en T (voir référence [14]),
• au niveau de l'orifice d'un dispositif microfluidique focalisant l'écoulement « MFFD » (i.e. « Microfluidic Flow Focusing Device », voir référence [15]),
• au travers de microcanaux structurés (cf. référence [16]), ou
• au travers de buses (voir référence [17]).

In the recent past, microfluidic encapsulation systems have also been developed which use emulsions that can be formed in particular:

• at a T-junction (see reference [14]),
• at the orifice of a microfluidic device focusing the flow "MFFD" (ie "Microfluidic Flow Focusing Device", see reference [15]),
• through structured microchannels (see reference [16]), or
• through nozzles (see reference [17]).

These encapsulation systems are the subject of numerous documents, among which the documents WO-2004/071638 , US-2007/0054119 , FR-A-2776535 , JP-A-2003 071261 and US-2006/0121122 and more particularly for the encapsulation of cells or clusters of cells and gelation of capsules formed, documents US-2006/0051329 , WO-2005/103106 and WO-2006/078841 .

The gelling step is carried out directly on the microsystem with serpentine or "H" microchannels, as described in the documents US-2006/0051329 and WO-2005/103106 .

The main disadvantage of these microfluidic encapsulation systems is the same as that mentioned in the introduction, which is to obtain a single size of capsules regardless of the size of the cell clusters. To the knowledge of the Applicant, only the device Wyman et al. (see reference [18] and the document US-2007/0009668 ) makes it possible to adapt the size of the capsule to the size of the cell clusters, such as islets of Langerhans, by wrapping them in capsules of constant thickness close to 20 μm, but independently of the size of the encapsulated islets. In the latter document, an aqueous phase is placed above an oil phase and, by adjusting the respective densities of these two phases, the islets are found at the water / oil interface. A sampling tube placed in the oil at a distance from the interface makes it possible to suck the aqueous phase and the islets in a fine jet, which, under the effect of the surface tension, breaks, leaving the Islet surface a thin hydrogel wrap of fixed thickness that is polymerized by UV irradiation. This device is however a macroscopic device, and not a microfluidic system.

An object of the present invention is to provide a microfluidic system that overcomes all the aforementioned drawbacks, which comprises a substrate in which is etched a microchannel network, which comprises a cell sorting unit and around which is sealed a cover of protection.

For this purpose, a microfluidic system according to the invention is such that the sorting unit comprises deflection means capable of separating, during their flow, preferably according to their size, clusters of slightly cohesive cells with a size of 20 μm. at 500 μm and from 20 to 10,000 cells each approximately, such as islands of Langerhans, at least two sorting microchannels arranged in parallel output of said unit being respectively designed to convey as many classes of sorted clusters to an encapsulation unit thereof also formed in said network .

By "size" of cell clusters or capsules coating them, in the present description is meant the diameter, in the case of a cluster or a substantially spherical capsule, or more generally the largest transverse dimension of this cluster or of this capsule (eg the long axis of an elliptical section in the approximation of an ellipsoid of revolution).

It should be noted that the microchannels dedicated to the sorting of the microsystem according to the invention are capable of separating these clusters of cells, such as islets of Langerhans, by their deviation, by their scale which is very different from that of known microfluidic systems only adapted to sorting. unique cells. In fact, the size of these islets varies in a known manner from 20 to 400 μm against 1 to 10 μm on average for a cell, and the islets must be handled with even greater precaution than single cells because of their fragility and their weak cohesion, which limits the range of shears applicable by the sorting unit.

Advantageously, said sorting unit may comprise at least one size sorting stage of said clusters which is designed to generate in said sort microchannels respectively at least two size categories for said sorted clusters.

It will be noted that the sorting stage (s) formed by a determined group of microchannels of the system according to the invention makes it possible to obtain as many size categories as desired (depending on the number of sorting microchannels scheduled in parallel), and in particular to adapt the size of the capsules formed following this sorting to the size of each category of sorted cell clusters.

It will also be noted that it is possible to couple several successive sorting stages by size (i.e. stages arranged one after the other) to optimize the final performance of the sorting unit.

According to one embodiment of the invention, said deflection means of said or each sorting stage are hydrodynamic to passive fluidic, preferably being of hydrodynamic focusing type, of deterministic lateral displacement type ("DLD") by means of an arrangement of deflection pads which comprises at least one microchannel of this stage, or of hydrodynamic filtration type by means of filtration microchannels arranged transversely to a main microchannel.

In a variant, these deflection means according to the invention of the or each sorting stage may be of the hydrodynamic type coupled to electrostatic or magnetic forces or to electromagnetic or acoustic waves.

According to another characteristic of the invention, an encapsulation unit, capable of automatically encapsulating said sorted clusters according to their category, is furthermore formed in said network in fluid communication with said sorting microchannels, this unit of encapsulation being able to form continuously around each sorted cluster a monolayer or multilayer capsule biocompatible, mechanically resistant and selective permeability.

This encapsulation unit may comprise a plurality of encapsulation subunits which are respectively arranged in parallel in communication with said sorting microchannels to form, for each size category of sorted clusters circulating therein, a capsule of predetermined size designed to wrap up each cluster of this category.

Advantageously, each encapsulation sub-unit may comprise a device for forming said capsules selected from the group consisting of "T" junction devices, microfluidic flow-focusing devices "MFDD", microchannel network devices. structured "MC array" and devices with network of microbuses "MN array".

In a variant, each encapsulation subunit may comprise a material exchanger between an aqueous phase comprising said clusters sorted within each category and a phase immiscible with this aqueous phase, for example an oily phase, this exchanger being designed to form the capsules by breaking the interface between these two phases due to overpressure.

According to another characteristic of the invention, said encapsulation unit may further comprise gelling means formed capsules, comprising a material exchanger consisting of microchannels and dedicated to the transfer of these capsules encapsulation phase containing them , for example of the oil-alginate type, to an aqueous gelling phase or not.

It will be noted that the microsystem according to the invention thus makes it possible to completely automate the encapsulation procedure of the cell clusters, in that the operator only has to fill the different reservoirs corresponding to the materials necessary for the encapsulation and recovers the capsules adapted to the size of the previously sorted clusters.

The microsystem therefore continuously and automatically performs the steps of sorting, capsule formation and gelation, and it can be adapted to both a simple encapsulation and a multilayer encapsulation. In the latter case, the encapsulation module is made more complex by integrating steps of rinsing the capsules and placing them in contact with other solutions of polymers or polycations.

Preferably, is furthermore formed in said microchannel network a microfluidic transfer module designed to transfer said sorted clusters of a culture medium containing them to an encapsulation phase intended to contain them in said encapsulation unit, this module transfer device being in fluid communication with each of said sorting microchannels and being designed to minimize the pressure losses in said sorting unit.

In fact, the islets intended for transplantation are preserved in a culture medium, but for encapsulation, they must be transferred. in a polymer solution (most often non-Newtonian fluid, high viscosity even at low shear). To automate the encapsulation procedure as much as possible, said transfer module is integrated in the microsystem between the sorting unit and the encapsulation unit so as to limit the pressure losses in this sorting unit, given that the fluidic resistance is proportional to the viscosity of the displaced solution.

This transfer module also has the advantage of reducing the total pressure in the microsystem, and thus to limit the risk of leakage when the pressures applied are too high.

According to another important feature of the invention, said microfluidic system furthermore advantageously comprises a module for coupling said sorting unit to said encapsulation unit, which is designed to maintain a laminar fluidic regime in these two units by directly communicating with each other. or selectively the encapsulation unit with the sorting unit.

It will be noted that no known microsystem has thus coupled the sorting step to that of encapsulation. However, this coupling is not easy to implement because the fluidics of the sorting unit can come to disturb that of the encapsulation unit. It is therefore necessary to model the overall pressure losses (i.e. fluid resistances) of the microchannels concerned, to maintain a laminar fluidic regime in these two units. This modeling is all the more complicated as encapsulation most often uses non-Newtonian polymers (e.g., alginate) whose viscosity depends on the shear applied to the fluid, which complicates the modeling of the overall system.

According to an exemplary embodiment of the invention, this coupling module consists of intermediate microchannels which respectively connect said sorting microchannels to said encapsulation unit and which have dimensions and geometry suitable for maintaining said laminar regime upstream and downstream.

The disadvantage of this coupling module according to this embodiment is that, in addition to the precise dimensional design which is For these intermediate microchannels, a large number of empty capsules may be formed in each encapsulation subunit, which may require at the outlet of the latter a final sorting between empty capsules and capsules containing sorted clusters.

According to another preferred embodiment of the invention, this coupling module comprises storage buffer microreservers sorted clusters, in each of which opens one of said sorting microchannels and which are each connected selectively to the encapsulation unit by an output microchannel which is intended to convey the sorted and concentrated clusters and which is equipped with a fluidic valve for example of the air bubble type or with a blocking gel which can be dissolved (preferably with alginate gel, in the case of the use of alginate for encapsulation), so that the opening and closing of the valve lowers and raises respectively the concentration of the clusters sorted in each microreservoir according to the number of capsules being formed in the encapsulation unit.

It should be noted that this preferential coupling module with a fluidic valve makes it possible to minimize the formation of empty capsules by this adjustment of the concentration in each microreservoir.

Advantageously, each buffer microreservoir may also be provided with a plurality of transverse output microchannel ends which are designed to allow the evacuation of the phase containing said clusters with the exception of the latter, when said valve is closed.

In general, it should be noted that the microfluidic systems according to the invention must be sterilizable because the capsules formed by the encapsulation unit must be able to be transplanted into an individual.

A process according to the invention for sorting out little cohesive clusters of cells ranging in size from 20 μm to 500 μm and from 20 to 10,000 cells, such as islets of Langerhans, consists in circulating these clusters in a network. microchannels of a microfluidic system of geometry adapted to the size and the number of these clusters to be separated, and to deflect them others according to one of their parameters, such as their size, so as to direct them to at least two sorting microchannels carrying in parallel as many categories of sorted clusters, for their encapsulation in the same system.

• une déviation hydrodynamique à fluidique passive, de préférence par focalisation hydrodynamique, par déplacement latéral déterministe (« DLD ») ou par filtration hydrodynamique, ou
• une déviation hydrodynamique couplée à des forces électrostatiques, magnétiques ou à des ondes électromagnétiques ou acoustiques.

Advantageously, at least one sorting stage is used for sorting said clusters to generate, in said sorting microchannels, respectively at least two size categories for said sorted clusters, each stage using:

• a hydrodynamic deviation to passive fluidic, preferably by hydrodynamic focusing, deterministic lateral shift ("DLD") or hydrodynamic filtration, or
• a hydrodynamic deviation coupled with electrostatic, magnetic forces or with electromagnetic or acoustic waves.

According to another characteristic of the invention, it is also possible to encapsulate in an automated manner these sorted clusters in parallel according to their category, forming continuously around each sorted cluster a biocompatible monolayer or multilayer capsule, mechanically resistant and selective permeability.

Advantageously, then, for each size category of sorted clusters, a capsule of predetermined size is formed which envelops each cluster of this category as closely as possible, preferably with a capsule size of approximately D _to +20 μm at D. _at +150 μm, preferably D _at +50 μm, for a class of clusters sorted according to a critical size less than a value D _a .

Preferably, these capsules are formed for each class of clusters sorted by a device selected from the group consisting of "T" junction devices, microfluidic flow-focusing devices "MFDD", microchannel network devices. structured "MC array" and devices with network of microbuses "MN array".

Alternatively, these capsules can be formed by exchange of material between an aqueous phase comprising the clusters sorted within each category and an immiscible phase with this aqueous phase, by oily example, the rupture of the interface between these two phases by an overpressure generating these capsules.

According to another characteristic of the invention, the capsules formed are then gelled by transfer of these capsules and the encapsulation phase containing them, for example of the oil-alginate type, to an aqueous gelling phase or not .

The polymer used for encapsulation may be, for example, an alginate hydrogel, the polymer most commonly used for encapsulation. However, the encapsulation according to the invention is not limited to this hydrogel and other encapsulation materials could be chosen, such as chitosan, carrageenans, agarose gels, polyethylene glycols (PEG), non-limiting, provided to adapt the encapsulation unit to the type of gelation that requires the chosen polymer.

Preferably, before each encapsulation, the sorted clusters are transferred from a culture medium containing them to the encapsulation phase intended to contain them, in order to minimize the losses during sorting.

Also preferentially, the method according to the invention further comprises a fluid coupling between the sorting and the encapsulation having the effect of maintaining a laminar fluidic regime in the corresponding microchannels, this coupling making said sorted bundles directly or selectively communicate with each other. the encapsulation phase.

As indicated above, this coupling can be achieved by means of intermediate microchannels of dimensions and geometry suitable for maintaining the laminar regime during sorting and encapsulation.

As a variant, this coupling is preferably carried out by regulating the concentration of each category of sorted clusters in a storage buffer storage buffer cluster communicating with one of said sorting microchannels and selectively connected by said fluidic valve to an outlet microchannel carrying the sorted and concentrated clusters, the opening and closing of this valve lowering and raising respectively the concentration of sorted clusters in the microreservoir according to the number of capsules being formed, to minimize the formation of capsules empty. This microreservoir is further advantageously provided with a plurality of transverse end microchannel ends designed to evacuate the single phase containing these clusters without them, when the valve is closed.

Advantageously, said clusters of cells sorted in the process of the invention are islets of Langerhans which are encapsulated with a size of capsules ranging from 70 μm to 200 μm for the islands sorted to a size of less than 50 μm, with a size of capsules up to 650 μm for larger islands sorted to a size of 500 microns for example.

A use according to the invention of a microfluidic system as presented above consists in sorting either cells, bacteria, organelles, liposomes or clusters of cells, preferably according to categories of interest via adhesion molecules. in the first case, or according to size categories in the case of cell clusters, then to encapsulate them continuously and automatically for each sorted category.

It will be noted that the invention is not limited solely to sorting by size and then to the encapsulation of cell clusters, but that it generally aims at coupling any encapsulation to a prior sorting of cells. cells, bacteria, organelles or liposomes within a heterogeneous population of these very different particles, so as to encapsulate only the cells / bacteria / organelles / liposomes of interest.

• la figure 1 est une vue schématique en coupe transversale d'un système microfluidique selon l'invention dans une première phase de son procédé de fabrication montrant l'oxydation du substrat,
• la figure 2 est une vue schématique en coupe transversale du système de la figure 1 dans une seconde phase de son procédé de fabrication montrant l'étalement d'une résine photosensible sur ce substrat oxydé,
• la figure 3 est une vue schématique en coupe transversale du système de la figure 2 dans une troisième phase de son procédé de fabrication montrant le résultat d'étapes suivantes de photolithographie et de gravure sèche, permettant de créer les microcanaux,
• la figure 4 est une vue schématique en coupe transversale du système de la figure 3 dans une quatrième phase de son procédé de fabrication montrant le résultat d'étapes de gravure profonde,
• la figure 5 est une vue schématique en coupe transversale du système de la figure 4 dans une cinquième phase de son procédé de fabrication montrant le résultat d'une étape de délaquage de la résine et de désoxydation par gravure humide,
• la figure 6 est une vue schématique en coupe transversale du système de la figure 5 dans une sixième phase de son procédé de fabrication montrant le résultat d'une étape de d'oxydation,
• la figure 7 est une vue schématique en coupe transversale du système de la figure 6 dans une septième phase de son procédé de fabrication montrant le résultat d'une étape de scellement d'un capot de protection afin de délimiter la section des microcanaux,
• la figure 8 est une vue schématique partielle de dessus d'un système microfluidique selon un exemple de réalisation de l'invention, montrant une unité de tri par filtration hydrodynamique et une unité d'encapsulation par des jonctions en T qui lui est couplée,
• la figure 9 est un cliché modélisant les lignes d'écoulement au sein d'un exemple d'unité de tri selon l'invention par focalisation hydrodynamique,
• la figure 10 est une vue schématique de dessus d'un microcanal d'une unité de tri selon l'invention qui est équipé de moyens de déviation par déplacement latéral déterministe (« DLD »),
• la figure 11 est une vue de détail du médaillon de la figure 10 montrant de manière symbolique un exemple de déviation de trajectoire obtenu par ces moyens de déviation,
• la figure 12 est un cliché modélisant les lignes d'écoulement au sein d'un autre exemple d'unité de tri selon l'invention par filtration hydrodynamique,
• la figure 13 est un cliché représentant schématiquement un agencement de microcanaux formant un module de transfert des îlots triés d'un milieu de culture vers une solution d'alginate utilisée pour l'encapsulation,
• la figure 14 est un diagramme à blocs illustrant quatre étages de tri respectivement couplés à quatre sous-unités d'encapsulation dans un exemple de mise en oeuvre du procédé de tri/ encapsulation selon l'invention,
• les figures 15 et 16 sont respectivement deux clichés représentant schématiquement une jonction en T et un dispositif focalisant de type « MFFD », chacun étant destiné à la formation d'une émulsion dans chaque sous-unité d'encapsulation selon l'invention,
• la figure 17 est une vue schématique d'un module de gélification inclus dans l'unité d'encapsulation selon l'invention, pour transférer les capsules formées d'une phase huileuse vers une phase aqueuse,
• la figure 17a est une vue schématique en coupe verticale d'un module de gélification selon une variante de la figure 17, qui peut être inclus dans l'unité d'encapsulation selon l'invention,
• la figure 17b est une vue schématique en coupe verticale d'un module de gélification selon une variante de la figure 17a, qui peut être inclus dans l'unité d'encapsulation selon l'invention,
• la figure 17c est une vue schématique partielle en coupe verticale d'une variante selon l'invention de l'élément séparateur prévu en sortie du module de gélification des figures 17a ou 17b,
• les figures 18 et 19 sont respectivement deux vues schématiques de modules de couplage selon un premier et un second exemples de l'invention, qui sont chacun reliés à un étage de tri et à une sous-unité correspondante d'encapsulation,
• la figure 20 est une vue schématique d'une unité d'encapsulation par fluidique passive selon un autre exemple de réalisation de l'invention, suite à un tri par taille effectué de préférence par déplacement latéral déterministe (« DLD »), et
• la figure 21 est une vue schématique d'une unité d'encapsulation selon l'invention illustrant notamment les étapes de formation de capsules à trois couches par un dispositif focalisant, et leur gélification.

Other advantages, characteristics and details of the invention will emerge from the additional description which will follow with reference to the accompanying drawings, given solely by way of example and in which:

• the figure 1 is a schematic cross-sectional view of a microfluidic system according to the invention in a first phase of its manufacturing process showing the oxidation of the substrate,
• the figure 2 is a schematic cross-sectional view of the system of the figure 1 in a second phase of its manufacturing process showing the spreading of a photosensitive resin on this oxidized substrate,
• the figure 3 is a schematic cross-sectional view of the system of the figure 2 in a third phase of his process of manufacturing showing the result of subsequent steps of photolithography and dry etching, to create the microchannels,
• the figure 4 is a schematic cross-sectional view of the system of the figure 3 in a fourth phase of its manufacturing process showing the result of deep etching steps,
• the figure 5 is a schematic cross-sectional view of the system of the figure 4 in a fifth phase of its manufacturing process showing the result of a step of delamination of the resin and deoxidation by wet etching,
• the figure 6 is a schematic cross-sectional view of the system of the figure 5 in a sixth phase of its manufacturing process showing the result of an oxidation step,
• the figure 7 is a schematic cross-sectional view of the system of the figure 6 in a seventh phase of its manufacturing process showing the result of a step of sealing a protective cover in order to delimit the section of the microchannels,
• the figure 8 is a partial schematic view from above of a microfluidic system according to an exemplary embodiment of the invention, showing a hydrodynamic filtration sorting unit and a unit of encapsulation by T-junctions coupled thereto,
• the figure 9 is a diagram modeling the flow lines within an example of a sorting unit according to the invention by hydrodynamic focusing,
• the figure 10 is a schematic view from above of a microchannel of a sorting unit according to the invention which is equipped with means of deflection by deterministic lateral displacement ("DLD"),
• the figure 11 is a detail view of the medallion of the figure 10 symbolically showing an example of trajectory deviation obtained by these deflection means,
• the figure 12 is a diagram modeling the flow lines in another example of a sorting unit according to the invention by hydrodynamic filtration,
• the figure 13 is a schematic diagram showing an arrangement of microchannels forming a transfer module of islands sorted from a culture medium to an alginate solution used for encapsulation,
• the figure 14 is a block diagram illustrating four sorting stages respectively coupled to four encapsulation subunits in an exemplary implementation of the sorting / encapsulation method according to the invention,
• the Figures 15 and 16 are respectively two plates schematically showing a T-junction and a focusing device of the "MFFD" type, each being intended for the formation of an emulsion in each encapsulation subunit according to the invention,
• the figure 17 is a schematic view of a gelling module included in the encapsulation unit according to the invention, for transferring the capsules formed from an oily phase to an aqueous phase,
• the figure 17a is a schematic view in vertical section of a gelling module according to a variant of the figure 17 , which may be included in the encapsulation unit according to the invention,
• the figure 17b is a schematic view in vertical section of a gelling module according to a variant of the figure 17a , which may be included in the encapsulation unit according to the invention,
• the figure 17c is a partial schematic view in vertical section of a variant according to the invention of the separating element provided at the outlet of the gelling module of the Figures 17a or 17b ,
• the figures 18 and 19 are respectively two schematic views of coupling modules according to first and second examples of the invention, which are each connected to a sorting stage and a corresponding sub-unit of encapsulation,
• the figure 20 is a schematic view of a passive fluidic encapsulation unit according to another embodiment of the invention, following a sorting by size preferably performed by deterministic lateral displacement ("DLD"), and
• the figure 21 is a schematic view of an encapsulation unit according to the invention illustrating in particular the steps of formation of three-layer capsules by a focusing device, and their gelation.

A microfluidic system 1 according to the invention can for example be made as follows, with reference to Figures 1 to 7 which report various steps based on known methods of microelectronics on silicon, ie lithography, deep etching, oxidation, "stripping" and sealing of a protective cover 2 on the substrate 3. technology on silicon has the advantage of being very precise (of the order of one micrometer) and not limiting both in the depths of etching and the width of the patterns. More specifically, the implementation protocol of microsystem 1 is as follows:

A deposit of silicon oxide 4 ( figure 1 ) is performed on the silicon substrate. Then a photosensitive resin 5 is deposited by spreading on the front face ( figure 2 ), whereupon the silicon oxide 4 is etched through the resin layer 5 by photolithography and dry etching of the silicon oxide 4, stopping on the silicon substrate 3 ( figure 3 ).

This substrate 3 is then etched at the desired depth of the microchannels by deep etching 6 ( figure 4 ), then the resin is "delacked" ( figure 5 ). The remaining thermal silicon oxide is then removed by deoxidation by means of wet etching ( figure 5 ), then a new layer of thermal oxide 7 is deposited ( figure 6 ).

The chips obtained are then cut and a protective cover 2 made of glass - or another transparent material to allow observation - is sealed, for example by anodic sealing or direct sealing ( figure 7 ).

Before mounting the microchannels or capillaries (not shown), a hydrophobic silanization surface treatment can also be performed.

The protocol described above is one of several manufacturing protocols that can be followed. Moreover, we could use for the substrate 3 a material other than silicon, for example a PDMS (polydimethylsiloxane) or else another elastomer, by molding on a "master" (ie matrix) previously prepared by photolithography for example. It will be noted that this manufacturing technique is well suited to the case where the microfluidic system comprises a coupling module between the sorting unit and that of encapsulation with fluidic valves, with reference to figures 18 and 19 .

The microfluidic system 101 according to the example of the invention illustrated in FIG. figure 8 comprises, on the one hand, a hydrostatic filtration cluster size unit 110 ending in four transverse sorting microchannels 111 to 114, and an encapsulation unit 120 subdivided into four encapsulation subunits 121 at 124 respectively coupled to these microchannels and conveying as many size categories of sorted clusters At.

The principle of this sorting unit 110 is illustrated in FIG. figure 12 and is based on a focus of cluster A to the wall. More precisely in relation with this figure 12 the fluidic resistances of the transverse microchannels 111 to 113 are adapted by choosing a suitable flow ratio between the main microchannel 115 and these transverse microchannels. As a result, clusters A can only penetrate one of the transverse microchannels 111 to 113, depending on their size and the respective fluidic resistances of these transverse microchannels, which are thus finely calculated to determine the size range of cluster A can penetrate into a particular microchannel 111, 112, 113 or 114.

The solution S for focusing the clusters A to the wall is injected into a secondary microchannel 116 communicating with the main microchannel 115 via branches 117 to 119, and this solution S may be the same as that containing the clusters A injected at the input E of the unit 110, being for example a culture medium or alginate.

• îlots At plus petits que 100 µm,
• îlots At de 100 à 200 µm,
• îlots At de 200 à 300 µm, et
• îlots At dépassant les 300 µm.

The sorting unit 110 thus makes it possible to sort clusters of A cells, such as islets of Langerhans, according to the following four categories:

• At islands smaller than 100 μm,
• At islands of 100 to 200 μm,
• At islands of 200 to 300 μm, and
• At islands greater than 300 μm.

As a variant of the figure 12 , it would be possible to use in the system the figure 8 the sorting unit 210 by hydrodynamic focusing of the figure 9 , in which is visible the entry of unsorted A clusters, a dynamic focusing device 211 using a focusing fluid S and, at the outlet of a deflection zone 212, a first sorting microchannel 213 conveying sorted clusters At ₁ deviated because they are the smallest and a second sorting microchannel 214 conveying the sorted clusters At ₂ as being the largest according to the hypothesis that the clusters of cells follow the flow lines on which their centers of inertia are positioned. An output microchannel 215 for a portion of the focusing fluid (without clusters) is further arranged at the output of this zone 212.

According to another variant of the figure 12 , it would also be possible to use in the system of the figure 8 sorting unit 310 by "DLD" of Figures 10 and 11 , using a network of pads 311 which is arranged in a predetermined manner inside a microchannel 312 and whose geometric characteristics impose a critical size Dc for cell clusters. Particles smaller than Dc are not deflected by the array of studs 312 and generally follow the fluid flow lines, whereas particles larger than Dc are deflected at each transverse row of studs 312 and thus separated from each other. smaller. It will be noted that several sort stages can be cascaded one after the other. This sorting unit 310 uses a focusing buffer solution F, which is injected at the same time as the solution containing the clusters A to be sorted.

As visible at figure 10 , at the output of this unit 310, on the one hand, the buffer solution F without clusters is recovered and, on the other hand, three categories of sorted clusters At ₁ , At ₂ and At ₃ which respectively correspond to this exemplary embodiment. to islets of Langerhans smaller than 200 μm, 200 to 300 μm and larger than 300 μm. So we have in this example put cascade two sorting stages of different geometrical characteristics, making it possible to obtain two critical sorting sizes Dc ₁ = 200 μm and Dc ₂ = 300 μm.

Returning to figure 8 , the four transverse sorting microchannels 111 to 114 conveying the sorted clusters At open respectively to the four encapsulation subunits 121 to 124, which are here of T-junction type each traversed by an oil H to form the capsules C , with reference to the figure 15 which shows in known manner the formation of an emulsion via the contact between the two phases of oil and alginate meeting in this junction. Alternatively, it would be possible to replace the T-junctions of the figure 8 by the focusing devices "MFFD" of the figure 16 making in this example converge two oily phases and an alginate phase.

• raccordés en amont d'une extrémité supérieure d'un pied vertical du H, un microcanal d'entrée 126 destiné à véhiculer des ions Ca²⁺ en solution aqueuse et, à l'autre extrémité inférieure de ce même pied, un dispositif d'encapsulation 127 de type « MFFD » à trois microcanaux convergents dont deux sont destinés à véhiculer la phase huileuse et le troisième de l'alginate pour former dans de l'huile les capsules C à base de Na-alginate, et
• raccordés en aval de l'extrémité supérieure de l'autre pied vertical du H, un microcanal de sortie 128 destiné à contenir un mélange de la solution aqueuse contenant les ions Ca²⁺ et ces capsules C transférées à base d'alginate et, à l'extrémité inférieure de cet autre pied, un microcanal 129 contenant la phase huileuse.

The figure 17 shows by way of example a possible structure of a gelling module 125 which is usable in each encapsulation subunit 121 to 124 of the figure 8 and which is capable of transferring the alginate-based capsules C from an oily phase to an aqueous phase to gel them. This module 125, for example generally in the shape of an "H", comprises:

• connected upstream of an upper end of a vertical leg of the H, an input microchannel 126 for conveying Ca ²⁺ ions in aqueous solution and at the other end of the same foot, a device for encapsulation 127 of the "MFFD" type with three convergent microchannels, two of which are intended to convey the oily phase and the third of the alginate to form in oil the Na-alginate capsules C, and
• connected downstream of the upper end of the other vertical leg of the H, an output microchannel 128 for containing a mixture of the aqueous solution containing the Ca ²⁺ ions and these transferred capsules C based on alginate and, the lower end of this other foot, a microchannel 129 containing the oily phase.

• deux entrées 136 et 137 comprenant :
  • * un microcanal horizontal d'entrée 136 destiné à l'acheminement d'une phase huileuse contenant les amas de cellules A_t encapsulés en amont, et
  • *un microcanal vertical d'entrée 137 communiquant avec le précédent et destiné à y injecter transversalement une phase aqueuse contenant un agent, tel que du calcium, apte à gélifier par polymérisation les capsules enrobant ces amas (à base d'un composé hydrophile, tel que l'alginate) ; et
• deux sorties 138 et 139 qui sont séparées l'une de l'autre par un séparateur ou « mur » 140 (réalisé par exemple en silicium, en verre ou en un élastomère tel qu'un PDMS, à titre non limitatif) et qui comprennent de part et d'autre de ce mur 140 :
  • * une sortie supérieure 138 destinée à véhiculer la phase aqueuse contenant les amas de cellules A_t encapsulés, par migration de ces amas de la phase huileuse vers la phase aqueuse supérieure du fait du caractère hydrophile du matériau (e.g. l'alginate) constituant les capsules, et
  • * une sortie inférieure 139 destinée à l'extraction de la phase huileuse.

The gelling module 135 illustrated in the variant of the figure 17a essentially comprises:

• two inputs 136 and 137 comprising:
  • an inlet horizontal microchannel 136 for conveying an oily phase containing the clusters of cells A _t encapsulated upstream, and
  • a vertical input microchannel 137 communicating with the preceding and intended to inject transversely an aqueous phase containing an agent, such as calcium, able to gel by polymerization the capsules coating these clusters (based on a hydrophilic compound, such as than alginate); and
• two outputs 138 and 139 which are separated from each other by a separator or "wall" 140 (made for example of silicon, glass or an elastomer such as a PDMS, non-limiting) and which comprise on both sides of this wall 140:
  • an upper outlet 138 intended to convey the aqueous phase containing the clusters of cells A _t encapsulated by migration of these clusters of the oily phase to the upper aqueous phase because of the hydrophilic nature of the material (eg alginate) constituting the capsules , and
  • a lower outlet 139 intended for the extraction of the oily phase.

The gelling module 145 illustrated in FIG. figure 17b differs only from that of the figure 17a in that, in the region of the horizontal inlet microchannel 136 which is the seat of the aforementioned migration by hydrophilic attraction, it is provided with an arrangement of trajectory-modifying pillars or pads 146 of the type used in the "DLD" devices. "(Ie with a spacing between two adjacent pillars 146 greater than the size of clusters A _t encapsulated) to amplify, by the effect of the deterministic lateral displacement in addition to this migration, the lateral displacement of clusters A _t encapsulated the oily phase to the upper aqueous phase.

As illustrated in figure 17c which presents an alternative embodiment of the separator 140 of the gelling module 135, 145 according to the Figures 17a or 17b it is advantageous to use a separator 150 in the form of a "double wall" to optimize the separation of the aqueous and oily phases. This separator 150 differs only from the previous one in that it is formed of two superimposed walls or partitions 151 and 152 separated from each other by a central interstitial channel 153, which makes it possible to recover at the output of the module 135 or 145 oily and aqueous phases which are each purer and eliminate through this interstitial channel 153 the central interface aqueous solution / oil. More precisely, the width of this channel 153 is designed so that the latter does not transport the clusters A _t encapsulated outside the gelling module 135, 145. It should be noted that this separator with a double partition 150 makes it possible in particular to reduce the traces of aqueous solution. in the oil, thus allowing reuse of the oil.

As a variant of these figures 17 , 17a, 17b and 17c it is possible, for example, to use, without limitation, a gelation module 225 such as that included in the Alginate-Poly-L-Lysine-Alginate three-layer encapsulation unit 220 according to US Pat. figure 21 where gelation is directly in 1-undecanol and not in aqueous phase.

As visible at this figure 21 the capsules are produced at an encapsulation device 221 of the "MFFD" type, and then gelled in the module 225 by introducing a 1-undecanol stream containing Cal ₂ . They are then transferred to the aqueous phase and rinsed at a first rinsing module 226 in the shape of "H".

The capsules are then placed in contact with a solution of PLL polycations (Poly-L-Lysine) in a serpentine channel 227, which makes it possible to adjust the incubation time of the capsules in this PLL solution. The capsules are subsequently rinsed in NaCl solution, to remove the unbound PLL in a second rinsing module 228, and the rinsing NaCl solution is then removed in the microchannels 229.

In the last step, the capsules are covered with an outer layer of alginate in an attachment module 230, for obtaining at the outlet of the unit 220 of the Alginate-PLL-Alginate three-layer capsules.

The figure 13 illustrates a usable structure of a cluster transfer module of cells (eg islets of Langerhans) from a culture medium to an alginate solution used for encapsulation, which can be advantageously included in a system. microfluidic according to the invention. The fluid resistances and the respective sizes of the microchannels forming this transfer module 20 are adjusted so that these sorted clusters are forced to flow into the main microchannel and thus pass from the culture medium to the alginate solution (or another polymer).

The figures 18 and 19 illustrate two preferred examples of coupling modules 30 and 40 which can each be coupled to one of the sorting stages 111 to 114 of the figure 8 and to each corresponding sub-unit of encapsulation 121 to 124 of this same figure 8 . Each coupling module 30, 40 is designed to maintain a laminar fluidic regime in both the sorting unit 110 and the encapsulation unit 120, selectively communicating these two units 110 and 120 to each other.

With reference to these two figures 18 and 19 , the corresponding coupling module 30, 40 comprises in both cases a storage buffer microreservoir 31, 41 sorted clusters, where a sorting microchannel 111 to 114 opens and which is connected selectively via a fluidic valve 32 , 42, to an encapsulation subunit 121 to 124 by an output microchannel 33, 50 for conveying the sorted and concentrated clusters when the valve 32, 42 is open. Each microreservoir 31, 41 is furthermore provided with a plurality of transverse microchannel output ends 34, 44 to allow the evacuation of the phase containing the clusters without the latter (eg the evacuation of the culture medium or the solution of alginate), when the valve 32, 42 is closed.

Closing the valve 32, 42 can store and especially concentrate the clusters so that their concentration in the encapsulation solution is sufficient to limit the number of empty capsules formed. The fine microchannels 34, 44 make it possible to ensure that the closure of the valve 32, 42 does not modify the fluid flow lines upstream in the corresponding sorting stage (the size of these microchannels 34, 44 is such that the clusters can not penetrate and are forced to concentrate in the microreservoir 31, 41).

More specifically with reference to the figure 18 In this example, an "air bubble" type valve 32 is used, the opening and closing of which are thermally controlled by means of a heating resistor 32a incorporated into a chip, in the following manner. When the air is kept at room temperature, the valve 32 is open. By increasing the temperature of the air contained in an activation chamber 32b of the valve, the pressure of the gas which is introduced into the outlet microchannel 33 is increased and blocks the passage of the fluid.

More specifically with reference to the figure 19 In this example, a valve 42 of the blocking gel type which can be dissolved and preferably alginate gel is used. Valve 42 is closed by forming an alginate gel 42a by contacting an alginate solution with Ca ²⁺ ions. The opening of the valve 42 corresponds to the dissolution of the alginate gel 42a with a solution of EDTA or any other chelator of Ca ²⁺ ions of sodium citrate or EGTA type. By controlling the relative pressures of the EDTA and Ca ^{2+ solutions} , the amount of each species is controlled so that if the EDTA is in excess, then all the Ca ²⁺ ions are chelated and the alginate gel 42a is dissolved by EDTA, and on the contrary free Ca ²⁺ ions allow the formation of the gel.

The position of the gel 42a is determined by the relative pressures of the alginate, Ca ²⁺ and EDTA phases. To prevent the microchannel 45 carrying the alginate from becoming clogged, a small amount of EDTA can be introduced at the same time as this alginate.

Once the cluster concentration step is complete and the alginate gel 42a dissolved, the circulation pressure of the EDTA (injected into two different microchannels 46 and 47 opposite to the output microchannel 43) and the circulation pressure Ca ²⁺ ions (injected into a microchannel 48 adjacent to a microchannel 49 conveying the culture medium) can be almost zero: only the alginate and this culture medium, completely harmless for the viability of the clusters, then circulate in the chamber 43. The latter is further provided with an output 50 for routing sorted and concentrated clusters to the corresponding encapsulation subunit 121 to 124, and an output 51 equipped with fine microchannel filtering 51a for the evacuation of only Ca ²⁺ ions.

It will be noted that the main advantage of this type of valve 42 is that there is no technological complication for incorporating it into the microsystem according to the invention.

The figure 20 schematically illustrates a variant of encapsulation unit 320 according to the invention, following a sorting by size performed by deterministic lateral displacement ("DLD"). The cells sorted At cells are encapsulated by passive fluidics, the encapsulation being generated at the rupture of the aqueous-oil interface when a local overpressure occurs.

• une première entrée 321 de phase aqueuse incluant les amas triés At en solution (e.g. dans du sérum physiologique, dans un milieu de culture ou dans de l'alginate, à titre non limitatif), cette entrée 321 définissant un microcanal horizontal 321a,
• une seconde entrée 322 d'une phase non miscible avec cette phase aqueuse (e.g. une huile, de l'undécanol, du « FC »), cette entrée 322 étant prévue à l'opposé et en contrebas de la première entrée 321,
• deux sorties opposées 323 et 324 pour la phase aqueuse introduite par la première entrée 321, qui sont prévues en dessous de cette dernière mais au-dessus de la seconde entrée 322 et qui sont reliées entre elles par deux microcanaux latéraux 323a et 324a (horizontaux) communiquant avec un microcanal vertical 325 prolongeant à angle droit le microcanal 321 a, et
• une sortie 326 pour l'évacuation de la phase non miscible ou huileuse contenant les amas At de cellules encapsulés qui est prévue à l'opposé et à la même hauteur que la seconde entrée 322 de cette phase non miscible, formant avec celle-ci un microcanal inférieur 327 d'encapsulation qui communique avec le microcanal vertical 325 de sorte à recevoir par gravité les amas provenant de la première entrée 321.

More specifically, this encapsulation unit 320 comprises:

• a first inlet 321 of aqueous phase including sorted clusters At in solution (eg in physiological saline, in a culture medium or in alginate, without limitation), this inlet 321 defining a horizontal microchannel 321a,
• a second inlet 322 of an immiscible phase with this aqueous phase (eg an oil, undecanol, "FC"), this inlet 322 being provided opposite and below the first inlet 321,
• two opposite outlets 323 and 324 for the aqueous phase introduced by the first inlet 321, which are provided below the latter but above the second inlet 322 and which are interconnected by two lateral microchannels 323a and 324a (horizontal) communicating with a vertical microchannel 325 extending at right angles to the microchannel 321a, and
• an outlet 326 for evacuating the immiscible or oily phase containing the clusters At of encapsulated cells which is provided opposite and at the same height as the second inlet 322 of this immiscible phase, forming therewith a lower microchannel 327 encapsulation which communicates with the vertical microchannel 325 so as to receive by gravity clusters from the first input 321.

It will be noted that this encapsulation unit 320, which is formed in three dimensions (in that the microfluidic inputs and outputs 321, 322, 323, 324 and 326 are not situated in the same plane), is capable of forming the capsules C not only by the aforementioned local overpressure resulting from the obstruction of the two lateral microchannels 323a and 324a, but also by the sedimentation force of the cell clusters due to gravity.

• îlots de taille inférieure à 100 µm triés en 111 et encapsulés en 121 par des capsules de 200 µm de diamètre ;
• îlots de taille comprise entre 100 et 200 µm triés en 112 et encapsulés en 122 par des capsules de 300 µm de diamètre ;
• îlots de taille comprise entre 200 et 300 µm triés en 113 et encapsulés en 123 par des capsules de 400 µm de diamètre ; et
• îlots de taille supérieure à 300 µm triés en 114 et encapsulés en 124 par des capsules de 500 µm de diamètre.

In conclusion and as illustrated by the example figure 14 , the method of sorting / encapsulation of the invention makes it possible to couple continuously and automatically a given number of encapsulation subunits 121-124 to as many sorting stages 111-114 of a sorting unit 110. preferably by size, via a corresponding number of coupling modules 30, 40. It is thus possible, for example, to sort islands of Langerhans into four categories respectively associated with related capsule sizes:

• islands of size less than 100 μm sorted into 111 and encapsulated in 121 by capsules of 200 μm in diameter;
• islands of size between 100 and 200 μm sorted at 112 and encapsulated at 122 by capsules of 300 μm in diameter;
• islands of size between 200 and 300 μm sorted into 113 and encapsulated in 123 by 400 μm diameter capsules; and
• islands of size greater than 300 μm sorted at 114 and encapsulated at 124 by capsules of 500 μm in diameter.

• une minimisation de la quantité de polymère à former autour des amas et donc du temps de réponse de ces derniers,
• une optimisation de la viabilité des amas encapsulés, notamment du fait que la diffusion de l'oxygène y est plus rapide, ce qui réduit les risques d'apparition de zones nécrosées lors des transplantations, et
• une minimisation du volume de capsules à transplanter, ce qui peut permettre l'implantation des capsules dans des zones plus propices à la revascularisation des tissus.

In this way, it is understood that the process according to the invention makes it possible to adapt as closely as possible to the size of the capsules formed, more sorting the clusters of cells, the size of the various categories of sorted clusters. It results advantageously:

• a minimization of the amount of polymer to form around the clusters and therefore the response time of the latter,
• an optimization of the viability of the encapsulated clusters, especially since the diffusion of oxygen is faster there, which reduces the risk of appearance of necrotic zones during transplants, and
• minimizing the volume of capsules to be transplanted, which may allow the implantation of the capsules in areas more conducive to tissue revascularization.

References cited:

1. 1. De Vos, Association between capsule diameter, adequacy of encapsulation, and survival of microencapsulated rat islet allografts. Transplantation, 1996. 62: p. 893-899 .
2. 2. Lehmann, R., Superiority of Small Islets in Human Islet Transplantation. Diabetes, 2007. 56: p. 594-603 .
3. 3. Zimmermann, Manufacture of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM-, and AFM-imaging. Biomaterials, 2003. 24: p. 2083-2096 .
4. 4. Goosen, Electrostatic droplet generation for encapsulation of somatic tissue: assessment of high-voltage power supply. Biotechnol.Prog., 1997. 13 (497-502). ).
5. 5. Seifert, Production of small, monodisperse alginate beads for cell immobilization. Biotechnol.Prog., 1997. 13: p. 562-568 .
6. 6. Huang, Continuous particles separation through deterministic lateral displacement. Science, 2004. 304 (987-990) ).
7. 7. Davis, Deterministic hydrodynamics: taking blood apart. PNAS, 2006. 103 (40): p. 14779-14784 .
8. 8. Inglis, Critical particle size for fractionation by deterministic lateral displacement. Lab on a chip, 2006. 6: p. 655-658 .
9. 9. Yamada, Microfluidic particle sorter employing flow splitting and recombining. Anal.Chem, 2006. 78: p. 1357-1362 .
10. 10. Yamada, Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics. Lab on a chip, 2005. 5: p. 1233-1239 .
11. 11. Yamada, Pinched flow fractionation: Continuous size separation of particles. Anal.Chem, 2004. 76: p. 5465-5471 .
12. 12. Takagi, Continuous particle separation in a microchannel having asymmetrically arranged multiple branches. Lab on a chip, 2005. 5: p. 778-784 .
13. 13. Website of the company Union Biometrica whose address is http://www.unionbio.com/applications/app_notes/app_islet.html
14. 14. Thorsen, Dynamic pattern formation in a vesicle-generating microfluidic device. Physical Review Letters, 2001. 86 (18): p. 4163-4166 .
15. 15. Anna, Training of dispersions using "for focusing" in microchannels. Applied Physics Letters, 2003. 82 (3): p. 364-366 .
16. 16. Suguira, Interfacial tension driven monodisperse droplet training from microfabricated channel array. Langmuir, 2001. 17: p. 5562-5566 .
17. 17. Sugiura, Size control of calcium alginate beads containing living cells using micronozzle array. Biomaterials, 2005. 26: p. 3327-3331 .
18. 18. Wyman, Immunoisolating pancreatic islets by encapsulation with selective withdrawal. Small, 2007. 3 (4): p. 683-690 .

Claims (23)

1. Microfluidic system (1, 101) comprising a substrate (3) in which an array of microchannels comprising a cell sorting unit (110, 210, 310) is etched and around which a protective cover (2) is bonded, characterized in that the sorting unit comprises deflection means (211, 311) capable of separating, during the flow thereof, relatively noncohesive cell clusters (A), each of size ranging from 20 µm to 500 µm and of 20 to 10 000 cells approximately, such as islets of Langerhans, at least two sorting microchannels (111 to 114) arranged in parallel at the outlet of said unit being respectively designed so as to transport as many categories of sorted clusters (At) to a unit for encapsulation (120, 220, 320) of the latter, also formed in said array, said sorting unit (110, 210, 310) preferably comprising at least one stage for size-sorting said clusters (A) which is designed so as to generate in said sorting microchannels (111 to 114) respectively at least two size categories for said sorted clusters (At).
2. Microfluidic system (1, 101) according to Claim 1, characterized in that said deflection means (211, 311) of said or of each sorting stage are passive fluidic hydrodynamic means, preferably being of hydrodynamic focusing type, of the type comprising deterministic lateral displacement (DLD) by means of an arrangement of deflection posts (311) that at least one microchannel (312) of this stage comprises, or else of the type comprising hydrodynamic filtration by means of filtration microchannels (111 to 114) arranged transversely to a main microchannel (115).
3. Microfluidic system (1, 101) according to Claim 1, characterized in that said deflection means of said or of each sorting stage are hydrodynamic means coupled to electrostatic or magnetic forces or to electromagnetic or acoustic waves.
4. Microfluidic system (1, 101) according to one of the preceding claims, characterized in that an encapsulation unit (120, 220, 320), capable of automated encapsulation of said sorted clusters (At) as a function of their category, is also formed in said array in fluidic communication with said sorting microchannels (111 to 114), said encapsulation unit being capable of continuously forming, around each sorted cluster, a biocompatible, mechanically strong, selectively permeable monolayer or multilayer capsule (C).
5. Microfluidic system (1, 101) according to Claim 4, characterized in that the encapsulation unit (120) comprises a plurality of encapsulation subunits (121 to 124) which are respectively arranged in parallel in communication with said sorting microchannels (111 to 114) so as to form, for each size category of sorted clusters (At) circulating therein, a capsule (C) of predetermined size designed so as to surround each cluster of this category as closely as possible.
6. Microfluidic system (1, 101) according to Claim 5, characterized in that each encapsulation subunit (121 to 124) comprises a device (127, 221) for forming said capsules (C), chosen from the group constituted of T-junctlon devices, microfluidic flow focusing devices (MFFDs), microchannel (MC) array devices and micronozzle (MN) array devices.
7. Microfluidic system (1, 101) according to Claim 5, characterized in that each encapsulation subunit (121 to 124) comprises an exchanger of material between an aqueous phase (321) comprising said sorted clusters (At) within each category and a phase (321) that is immiscible with this aqueous phase, for example an oil phase, this exchanger being designed so as to form the capsules (C) by rupturing of the interface between these two phases due to an increased pressure.
8. Microfluidic system (1, 101) according to one of Claims 4 to 7, characterized in that said encapsulation unit (120, 220) also comprises means (125, 135, 145, 225) for gelling the capsules (C) formed, comprising an exchanger of material constituted of microchannels and dedicated to the transfer of these capsules from an encapsulation phase containing them, for example of oil-alginate type, to an aqueous or nonaqueous gelling phase.
9. Microfluidic system (1, 101) according to one of Claims 4 to 7, characterized in that there is also formed in said microchannel array a microfluidic transfer module (20) designed so as to transfer said sorted clusters (At) from a culture medium containing them to an encapsulation phase intended to contain them in said encapsulation unit (120, 220, 320), this transfer module being in fluidic communication with each of said sorting microchannels (111 to 114) and being designed so as to minimize the pressure losses in said sorting unit (110, 210, 310).
10. Microfluidic system (1, 101) according to one of Claims 4 to 9, characterized in that it also comprises a module (30, 40) for coupling said sorting unit (110, 210, 310) to said encapsulation unit (120, 220, 320), which is designed so as to maintain laminar fluidic conditions in these two units by causing the encapsulation unit to communicate directly or else selectively with the sorting unit.
11. Microfluidic system (1, 101) according to Claim 10, characterized in that said coupling module is constituted of intermediate microchannels which respectively connect said sorting microchannels (111 to 114) to said encapsulation unit (120, 220, 320) and which have dimensions and a geometry suitable for maintaining said laminar conditions upstream and downstream.
12. Microfluidic system (1, 101) according to Claim 10, characterized in that said coupling module (30, 40) comprises buffer microreservoirs (31, 41) for storing said sorted clusters (At), opening out into each of which is one of said sorting microchannels (111 to 114) and which are each connected selectively to said encapsulation unit (120, 220, 320) via an outlet microchannel (33, 43) which is intended to transport said sorted and concentrated clusters and which is equipped with a fluidic valve (32, 42), for example of air bubble type or of the type comprising a dissolvable blocking gel, such that the opening and the closing of this valve lowers and raises, respectively, the concentration of said sorted clusters in each microreservoir as a function of the number of capsules (C) undergoing formation in said encapsulation unit, each microreservoir (31, 41) also preferably having a plurality of fine transverse outlet microchannels (34, 44) which are designed so as to allow expulsion of the phase containing said clusters (At) with the exception of the latter, when said valve (32, 42) is closed.
13. Method for sorting relatively noncohesive cell clusters (A) of size ranging from 20 µm to 500 µm and of 20 to 10 000 cells approximately, such as islets of Langerhans, characterized in that it consists in circulating these clusters in a microchannel array of a microfluidic system (1, 101) having a geometry suitable for the size and for the number of these clusters to be separated, and in deflecting them from one another according to one of their parameters, such as their size, in such a way as to direct them to at least two sorting microchannels (111 to 114) transporting, in parallel, as many categories of sorted clusters (At), with a view to the encapsulation thereof in this same system, and in that use is preferably made of at least one stage for size-sorting said clusters (A) in order to generate in said sorting microchannels (111 to 114) respectively at least two size categories for said sorted clusters (At), each stage using:

    - passive fluidic hydrodynamic deviation, preferably by hydrodynamic focusing, by deterministic lateral displacement (DLD) or by hydrodynamic filtration, or

    - hydrodynamic deviation coupled to electrostatic or magnetic forces or to electromagnetic or acoustic waves.
14. Sorting method according to Claim 13, characterized in that said sorted clusters (At) are also encapsulated, in an automated manner, in parallel, as a function of their category, by continuously forming around each sorted cluster a biocompatible, mechanically strong, selectively permeable monolayer or multilayer capsule (C), this capsule being, for example, based on an alginate hydrogel.
15. Method for sorting and continuous encapsulation according to Claim 14, characterized in that there is formed, for each size category of sorted clusters (At), a capsule (C) of predetermined size which surrounds each cluster of this category as closely as possible, preferably with a capsule size of approximately D_a+20 µm to D_a+150 µm for a category of sorted clusters according to a critical size less than a value D_a.
16. Method for sorting and continuous encapsulation according to Claim 14 or 15, characterized in that said capsules (C) are formed for each category of sorted clusters (At) by means of a device (127, 221) chosen from the group constituted of T-junction devices, microfluidic flow focusing devices, MFFDs, microchannel array, MC array, devices and micronozzle array, MN array, devices, and in that said capsules (C) are preferably formed by exchange of material between an aqueous phase (321) comprising said sorted clusters (At) within each category and a phase (322) that is immiscible with this aqueous phase, for example an oily phase, the rupturing of the interface between these two phases by an increased pressure generating these capsules.
17. Method for sorting and continuous encapsulation according to one of Claims 14 to 16, characterized in that the capsules (C) formed are then gelled by transferring these capsules and the encapsulation phase containing them, for example of oil-alginate type, to an aqueous or nonaqeuous gelling phase.
18. Method for sorting and continuous encapsulation according to one of Claims 14 to 17, characterized in that, before each encapsulation, said sorted clusters (At) are transferred from a culture medium containing them to the encapsulation phase intended to contain them, so as to minimize the pressure losses during the sorting.
19. Method for sorting and continuous encapsulation according to one of Claims 14 to 18, characterized in that it also comprises fluidic coupling between the sorting and the encapsulation, which has the effect of maintaining laminar fluidic conditions in the corresponding microchannels, this coupling causing said sorted clusters (At) to communicate directly or else selectively with the encapsulation phase.
20. Method for sorting and continuous encapsulation according to Claim 19, characterized in that this coupling is carried out by means of fine intermediate microchannels which have dimensions and a geometry suitable for maintaining the laminar conditions during the sorting and during the encapsulation.
21. Method for sorting and continuous encapsulation according to Claim 19, characterized in that this coupling is carried out by adjusting the concentration of each category of sorted clusters (At) in a buffer microreservoir (31, 41) for storing these clusters which is in communication with one of said sorting microchannels (111 to 114) and selectively connected, via a fluidic valve (32, 42), to an outlet microchannel (33, 43) transporting the sorted and concentrated clusters, the opening and the closing of this valve lowering and raising, respectively, the concentration of the sorted clusters in the microreservoir as a function of the number of capsules (C) undergoing formation, so as to minimize the formation of empty capsules, this microreservoir being provided with a plurality of fine transverse outlet microchannels (34, 44) designed so as to expel the aqueous phase containing these clusters with the exception of the latter, when the valve is closed.
22. Method for sorting and continuous encapsulation according to one of Claims 14 to 21, characterized in that said cell clusters (At) are islets of Langerhans and are encapsulated with a capsule (C) size ranging from 70 µm to 200 µm for the islets sorted according to a size less than 50 µm, with a capsule size that can reach 650 µm for the largest islets sorted.
23. Use of a microfluidic system (1, 101) according to one of Claims 1 to 12, for sorting either cells, bacteria, organelles or liposomes, or cell clusters (At), preferably according to categories of interest via adhesion molecules in the first case, or else according to size categories in the case of cell clusters, and then for encapsulating them continuously and in an automated manner for each category sorted.

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