EP2119503A2 - Microfluid system and method for sorting clusters of cells and continuously encapsulating them once they are sorted - Google Patents
Microfluid system and method for sorting clusters of cells and continuously encapsulating them once they are sorted Download PDFInfo
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- EP2119503A2 EP2119503A2 EP09290311A EP09290311A EP2119503A2 EP 2119503 A2 EP2119503 A2 EP 2119503A2 EP 09290311 A EP09290311 A EP 09290311A EP 09290311 A EP09290311 A EP 09290311A EP 2119503 A2 EP2119503 A2 EP 2119503A2
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- encapsulation
- sorting
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- sorted
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/2525—Stabilizing or preserving
Definitions
- 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).
- islets of Langerhans cluster of fragile cells located in the pancreas and consisting of several cell types, including ⁇ 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 ⁇ cells.
- 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 ⁇ m to 400 ⁇ m for islets of Langerhans against about ten ⁇ m for a single cell) and also because of its size. its weak cohesion (which imposes weak shears in the microfluidic sorting system used).
- 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 .
- 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.
- a microfluidic system 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 ⁇ m. at 500 ⁇ m 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 .
- 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).
- 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.
- the size of these islets varies in a known manner from 20 to 400 ⁇ m against 1 to 10 ⁇ m 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.
- 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.
- 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.
- 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.
- DLD deterministic lateral displacement type
- 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.
- 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.
- 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”.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- a polymer solution most often non-Newtonian fluid, high viscosity even at low shear.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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)
- 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.
- 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.
- 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 ⁇ m to 500 ⁇ m 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.
- 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 ⁇ m at D. at +150 ⁇ m, preferably D at +50 ⁇ m, for a class of clusters sorted according to a critical size less than a value D a .
- 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”.
- 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.
- 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.
- 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.
- 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.
- 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.
- this coupling can be achieved by means of intermediate microchannels of dimensions and geometry suitable for maintaining the laminar regime during sorting and encapsulation.
- 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.
- 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 ⁇ m to 200 ⁇ m for islands sorted to a size of less than 50 ⁇ m, with a size of capsules up to 650 ⁇ m 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.
- 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.
- 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 ).
- a hydrophobic silanization surface treatment can also be performed.
- the protocol described above is one of several manufacturing protocols that can be followed.
- 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.
- PDMS polydimethylsiloxane
- master ie matrix
- the microfluidic system 101 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- FIG 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.
- 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.
- 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.
- 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.
- PLL polycations Poly-L-Lysine
- 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.
- 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.
- a cluster transfer module of cells eg islets of Langerhans
- 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.
- 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).
- 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.
- the valve 32 is open.
- the pressure of the gas which is introduced into the outlet microchannel 33 is increased and blocks the passage of the fluid.
- 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.
- 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.
- 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.
- valve 42 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.
- 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.
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Abstract
Description
La présente invention concerne un système microfluidique et un procédé pour le tri d'amas de cellules, tels que des îlots de Langerhans, et pour l'encapsulation en continu et de manière automatisée des amas une fois triés dans des capsules de tailles adaptées à celles de ces amas triés. L'invention s'applique en particulier au couplage entre tri et encapsulation de tels amas de cellules, mais également d'une manière plus générale de cellules, de bactéries, d'organelles ou de liposomes, notamment.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.
L'encapsulation cellulaire est une technique qui consiste à immobiliser des cellules ou des amas de cellules dans des microcapsules, de façon à les protéger des attaques du système immunitaire lors d'une transplantation. La porosité des capsules doit permettre l'entrée des molécules de faible poids moléculaire essentielles au métabolisme des cellules encapsulées, telles que des molécules de nutriments, d'oxygène, etc., tout en empêchant l'entrée de substances de poids moléculaire plus élevé comme les anticorps ou les cellules du système immunitaire. Cette perméabilité sélective des capsules est ainsi conçue pour assurer l'absence de contact direct entre les cellules encapsulées du donneur et celles du système immunitaire du receveur de la transplantation, ce qui permet de limiter les doses de traitement immunosuppresseur utilisées lors de la transplantation (ce traitement présentant des effets secondaires lourds).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).
Parmi les multiples applications de l'encapsulation, on peut citer celle des îlots de Langerhans, amas de cellules fragiles situés dans le pancréas et constitués de plusieurs types cellulaires dont les cellules β qui régulent la glycémie dans le corps par production de l'insuline. L'encapsulation de ces îlots est une alternative aux thérapies cellulaires classiques (e.g. transplantation de pancréas ou d'îlots) utilisées pour soigner le diabète insulinodépendant, maladie auto-immune dans laquelle le système immunitaire détruit ses propres cellules β productrices d'insuline.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.
Les capsules produites doivent répondre à certains critères, dont la biocompatibilité, la résistance mécanique et la perméabilité sélective, en particulier. Un autre critère essentiel est la taille des capsules, car en l'ajustant au mieux à la taille des amas de cellules (voir référence [1]):
- 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 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. .
Si les propriétés de biocompatibilité, de résistance mécanique ou de perméabilité sélective semblent bien acquises d'après la littérature, il n'en est pas de même pour la taille des capsules qui est particulièrement problématique pour l'encapsulation des îlots de Langerhans. En effet, dans tous les documents connus de la Demanderesse à ce jour, la taille des capsules formées autour de ces îlots est fixe et en moyenne de l'ordre de 600 à 800 µm, alors que ces îlots ont une taille variant de 20 à 400 µm seulement. Une taille de capsules fixe et identique quelle que soit la taille de l'îlot pose donc problème, d'autant plus que des études récentes ont montré que les îlots les plus performants sont les plus petits (voir référence [2]).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]).
Les principales méthodes d'encapsulation connues utilisent au choix :
- 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.
- 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 principaux inconvénients de ces techniques sont :
- 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 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.
On a développé récemment des systèmes microfluidiques adaptés au tri par taille de bactéries, de cellules, d'organelles, de virus, d'acides nucléiques ou même de protéines, parmi lesquels on peut citer :
- 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 US-A-2007/0026381 - les systèmes réalisant un tri par filtration hydrodynamique (voir références [9, 10] et les documents
JP-A-2007 021465 JP-A-2006 263693 JP-A-2004 154747 - 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 - 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 WO-A-2006/079007 - 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 WO-A-02/40874
- 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 US-2007/0026381 - systems performing sorting by hydrodynamic filtration (see references [9, 10] and documents
JP-A-2007 021465 JP-A-2006 263693 JP-A-2004 154747 - simpler sorting systems using only the deviation of the flow lines (see references [11, 12] and for example the document
WO-2006/102258 - the sorting systems using filters that allow either to pass molecules smaller than a critical value (see document
US-2005/0133480 WO-2006/079007 - sorting systems where the microfluidic is coupled to an external field, such as optical fluorescence or absorbance measurements (see the documents
WO-2002/023163 WO-A-02/40874
Un inconvénient majeur de tous les systèmes microfluidiques de tri présentés dans ces documents est qu'ils ne sont pas du tout adaptés au tri d'amas de cellules, tels que des îlots de Langerhans ou d'autres amas peu cohésifs de tailles similaires. En effet et comme expliqué précédemment, chacun de ces amas se comporte bien différemment d'une cellule du fait de sa taille (de 20 µm à 400 µm pour des îlots de Langerhans contre une dizaine de µm pour une cellule unique) et également du fait de sa faible cohésion (qui impose des cisaillements faibles dans le système microfluidique de tri utilisé).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).
Le seul système connu de la Demanderesse pour réaliser le tri de tels amas de cellules, est le cytomètre en flux de dénomination « COPAS » qui est commercialisé par la société Union Biometrica. Ce système, qui n'est pas de type microfluidique, réalise le tri par taille des amas en mesurant leurs temps de vols respectifs dans le faisceau d'un rayonnement laser (voir référence [13]).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]).
On a également développé dans un passé récent des systèmes microfluidiques d'encapsulation qui utilisent des émulsions pouvant être notamment formées :
- 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]).
- 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]).
Ces systèmes d'encapsulation font l'objet de nombreux documents, parmi lesquels les documents
L'étape de gélification est effectuée directement sur le microsystème avec des microcanaux en serpentin ou en « H », comme décrit dans les documents
Le principal inconvénient de ces systèmes microfluidiques d'encapsulation est le même que celui précité en introduction, qui est l'obtention d'une seule taille de capsules quelle que soit la taille des amas de cellules. A la connaissance de la Demanderesse, seul le dispositif de Wyman et al. (voir référence [18] et le document
Un but de la présente invention est de proposer un système microfluidique remédiant à l'ensemble des inconvénients précités, qui comporte un substrat dans lequel est gravé un réseau de microcanaux, qui comprend une unité de tri de cellules et autour duquel est scellé un capot de protection.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.
A cet effet, un système microfluidique selon l'invention est tel que l'unité de tri comporte des moyens de déviation aptes à séparer lors de leur écoulement, de préférence selon leur taille, des amas de cellules peu cohésifs de taille variant de 20 µm à 500 µm et de 20 à 10 000 cellules chacun environ, tels que des îlots de Langerhans, au moins deux microcanaux de tri agencés en parallèle en sortie de ladite unité étant respectivement conçus pour véhiculer autant de catégories d'amas triés vers une unité d'encapsulation de ces derniers également formée dans ledit réseau.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 .
Par « taille » des amas de cellules ou des capsules les enrobant, on entend dans la présente description le diamètre, dans le cas d'un amas ou d'une capsule sensiblement sphérique, ou plus généralement la plus grande dimension transversale de cet amas ou de cette capsule (e.g. le grand axe d'une section elliptique dans l'approximation d'un ellipsoïde de révolution).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).
On notera que les microcanaux dédiés au tri du microsystème selon l'invention sont aptes à séparer par déviation ces amas de cellules, tels que des îlots de Langerhans, de par leur échelle qui est bien différente de celle des systèmes microfluidiques connus seulement adaptés au tri de cellules uniques. En effet, la taille de ces îlots varie de manière connue de 20 à 400 µm contre 1 à 10 µm en moyenne pour une cellule, et les îlots doivent être manipulés avec encore plus de précaution que des cellules uniques à cause de leur fragilité et de leur faible cohésion, ce qui limite la gamme de cisaillements applicable par l'unité de tri.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.
Avantageusement, ladite unité de tri peut comprendre au moins un étage de tri par taille desdits amas qui est conçu pour générer dans lesdits microcanaux de tri respectivement au moins deux catégories de tailles pour lesdits amas triés.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.
On notera que le(s) étage(s) de tri par taille formé(s) par un groupe déterminé de microcanaux du système selon l'invention permet(tent) d'obtenir autant de catégories de tailles que souhaité (en fonction du nombre de microcanaux de tri prévus en parallèle), et en particulier d'adapter la taille des capsules formées suite à ce tri à la taille de chaque catégorie d'amas triés de cellules.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.
On notera également qu'il est possible de coupler plusieurs étages successifs de tri par taille (i.e. des étages agencés les uns à la suite des autres) pour optimiser la performance finale de l'unité de tri.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.
Selon un mode de réalisation de l'invention, lesdits moyens de déviation dudit ou de chaque étage de tri sont hydrodynamiques à fluidique passive, étant de préférence de type à focalisation hydrodynamique, de type à déplacement latéral déterministe (« DLD ») au moyen d'un arrangement de plots de déviation que comporte au moins un microcanal de cet étage, ou bien de type à filtration hydrodynamique au moyen de microcanaux de filtration agencés transversalement à un microcanal principal.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.
En variante, ces moyens de déviation selon l'invention du ou de chaque étage de tri peuvent être de type hydrodynamiques couplés à des forces électrostatiques, magnétiques ou à des ondes électromagnétiques ou acoustiques.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.
Selon une autre caractéristique de l'invention, une unité d'encapsulation, apte à encapsuler de manière automatisée lesdits amas triés en fonction de leur catégorie, est en outre formée dans ledit réseau en communication fluidique avec lesdits microcanaux de tri, cette unité d'encapsulation étant apte à former en continu autour de chaque amas trié une capsule monocouche ou multicouches biocompatible, mécaniquement résistante et à perméabilité sélective.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.
Cette unité d'encapsulation peut comprendre une pluralité de sous-unités d'encapsulation qui sont respectivement agencées en parallèle en communication avec lesdits microcanaux de tri pour former, pour chaque catégorie de taille d'amas triés y circulant, une capsule de taille prédéterminée conçue pour envelopper au plus près chaque amas de cette catégorie.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.
Avantageusement, chaque sous-unité d'encapsulation peut comporter un dispositif de formation desdites capsules choisi dans le groupe constitué par les dispositifs à jonction en « T », les dispositifs microfluidiques de focalisation d'écoulement « MFDD », les dispositifs à réseau de microcanaux structurés « MC array » et les dispositifs à réseau de microbuses « MN array ».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".
En variante, chaque sous-unité d'encapsulation peut comporter un échangeur de matière entre une phase aqueuse comprenant lesdits amas triés au sein de chaque catégorie et une phase non miscible avec cette phase aqueuse, par exemple huileuse, cet échangeur étant conçu pour former les capsules par rupture de l'interface entre ces deux phases due à une surpression.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.
Selon une autre caractéristique de l'invention, ladite unité d'encapsulation peut comprendre en outre des moyens de gélification des capsules formées, comprenant un échangeur de matière constitué de microcanaux et dédié au transfert de ces capsules d'une phase d'encapsulation les contenant, par exemple de type huile-alginate, vers une phase de gélification aqueuse ou non.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.
On notera que le microsystème selon l'invention permet ainsi d'automatiser entièrement la procédure d'encapsulation des amas de cellules, en ce sens que l'opérateur n'a plus qu'à remplir les différents réservoirs correspondant aux matériaux nécessaires à l'encapsulation et récupère en sortie les capsules adaptées à la taille des amas préalablement triés.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.
Le microsystème réalise donc de manière continue et automatisée les étapes de tri, de formation des capsules et de gélification, et il peut être adapté aussi bien à une simple encapsulation qu'à une encapsulation multicouches. Dans ce dernier cas, le module d'encapsulation est complexifié par intégration d'étapes de rinçage des capsules et de mise en contact avec d'autres solutions de polymères ou de polycations.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.
De préférence, est en outre formé dans ledit réseau de microcanaux un module microfluidique de transfert conçu pour transférer lesdits amas triés d'un milieu de culture les contenant vers une phase d'encapsulation destinée à les contenir dans ladite unité d'encapsulation, ce module de transfert étant en communication fluidique avec chacun desdits microcanaux de tri et étant conçu pour minimiser les pertes de charge dans ladite unité de tri.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.
En effet, les îlots destinés à la transplantation sont conservés dans un milieu de culture, mais pour l'encapsulation, ils doivent être transférés dans une solution de polymère (fluide le plus souvent non newtonien, de viscosité élevée même à faible cisaillement). Pour automatiser au maximum la procédure d'encapsulation, ledit module de transfert est intégré au microsystème, entre l'unité de tri et celle d'encapsulation de façon à limiter les pertes de charges dans cette unité de tri, compte tenu du fait que la résistance fluidique est proportionnelle à la viscosité de la solution déplacée.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.
Ce module de transfert présente en outre l'avantage de diminuer la pression totale dans le microsystème, et donc de limiter les risques de fuites lorsque les pressions appliquées sont trop élevées.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.
Selon une autre caractéristique importante de l'invention, ledit système microfluidique comprend en outre avantageusement un module de couplage de ladite unité de tri à ladite unité d'encapsulation, qui est conçu pour maintenir un régime fluidique laminaire dans ces deux unités en faisant communiquer directement ou bien sélectivement l'unité d'encapsulation avec l'unité de tri.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.
On notera qu'aucun microsystème connu n'a ainsi couplé l'étape de tri à celle d'encapsulation. Or, ce couplage n'est pas aisé à mettre en oeuvre, car la fluidique de l'unité de tri peut venir perturber celle de l'unité d'encapsulation. Il est donc nécessaire de modéliser les pertes de charges (i.e. les résistances fluidiques) globales des microcanaux concernés, pour conserver un régime fluidique laminaire dans ces deux unités. Cette modélisation est d'autant plus compliquée que l'encapsulation utilise le plus souvent des polymères non newtoniens (e.g. l'alginate) dont la viscosité dépend du cisaillement appliqué au fluide, ce qui complexifie la modélisation du système global.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.
Selon un exemple de réalisation de l'invention, ce module de couplage est constitué de microcanaux intermédiaires qui relient respectivement lesdits microcanaux de tri à ladite unité d'encapsulation et qui présentent des dimensions et une géométrie adaptées pour le maintien dudit régime laminaire en amont et en aval.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.
L'inconvénient de ce module de couplage selon cet exemple de réalisation est que, outre la conception dimensionnelle précise qui est requise pour ces microcanaux intermédiaires, il peut y avoir formation d'un grand nombre de capsules vides dans chaque sous-unité d'encapsulation, ce qui peut nécessiter en sortie de cette dernière un tri final entre capsules vides et capsules contenant des amas triés.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.
Selon un autre exemple préférentiel de réalisation de l'invention, ce module de couplage comprend des microréservoirs tampon de stockage des amas triés, dans chacun desquels débouche l'un desdits microcanaux de tri et qui sont chacun reliés sélectivement à l'unité d'encapsulation par un microcanal de sortie qui est destiné à véhiculer les amas triés et concentrés et qui équipé d'une vanne fluidique par exemple de type à bulle d'air ou à gel bloquant pouvant être dissous (de préférence à gel d'alginate, dans le cas de l'utilisation d'alginate pour l'encapsulation), de sorte que l'ouverture et la fermeture de la vanne abaisse et élève respectivement la concentration des amas triés dans chaque microréservoir en fonction du nombre de capsules en cours de formation dans l'unité d'encapsulation.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.
On notera que ce module de couplage préférentiel à vanne fluidique permet de minimiser la formation de capsules vides par ce réglage de la concentration dans chaque microréservoir.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.
Avantageusement, chaque microréservoir tampon peut être en outre pourvu d'une pluralité de fins microcanaux transverses de sortie qui sont conçus pour permettre l'évacuation de la phase contenant lesdits amas à l'exception de ces derniers, lorsque ladite vanne est fermée.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.
D'une manière générale, on notera que les systèmes microfluidiques selon l'invention doivent être stérilisables, car les capsules formées par l'unité d'encapsulation doivent pouvoir être transplantées chez un individu.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.
Un procédé selon l'invention pour le tri d'amas peu cohésifs de cellules de taille variant de 20 µm à 500 µm et de 20 à 10 000 cellules environ, tels que des îlots de Langerhans, consiste à faire circuler ces amas dans un réseau de microcanaux d'un système microfluidique de géométrie adaptée à la taille et au nombre de ces amas à séparer, et à les dévier les uns des autres selon l'un de leurs paramètres, tel que leur taille, de manière à les diriger vers au moins deux microcanaux de tri véhiculant en parallèle autant de catégories d'amas triés, en vue de leur encapsulation dans ce même système.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.
Avantageusement, l'on utilise au moins un étage de tri par taille desdits amas pour générer dans lesdits microcanaux de tri respectivement au moins deux catégories de tailles pour lesdits amas triés, chaque étage utilisant :
- 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.
- 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.
Selon une autre caractéristique de l'invention, l'on peut encapsuler en outre de manière automatisée ces amas triés en parallèle en fonction de leur catégorie, en formant en continu autour de chaque amas trié une capsule monocouche ou multicouches biocompatible, mécaniquement résistante et à perméabilité sélective.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.
Avantageusement, l'on forme alors, pour chaque catégorie de taille d'amas triés, une capsule de taille prédéterminée enveloppant au plus près chaque amas de cette catégorie, de préférence avec une taille de capsule d'environ Da+20 µm à Da+150 µm, préférentiellement Da+50 µm, pour une catégorie d'amas triés selon une taille critique inférieure à une valeur Da.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 .
De préférence, on forme ces capsules pour chaque catégorie d'amas trié par un dispositif choisi dans le groupe constitué par les dispositifs à jonction en « T », les dispositifs microfluidiques de focalisation d'écoulement « MFDD », les dispositifs à réseau de microcanaux structurés « MC array » et les dispositifs à réseau de microbuses « MN array ».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".
En variante, on peut former ces capsules par échange de matière entre une phase aqueuse comprenant les amas triés au sein de chaque catégorie et une phase non miscible avec cette phase aqueuse, par exemple huileuse, la rupture de l'interface entre ces deux phases par une surpression générant ces capsules.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.
Selon une autre caractéristique de l'invention, l'on gélifie ensuite les capsules formées, par un transfert de ces capsules et de la phase d'encapsulation les contenant, par exemple de type huile-alginate, vers une phase de gélification aqueuse ou non.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 .
Le polymère utilisé pour l'encapsulation peut être par exemple un hydrogel d'alginate, polymère le plus couramment utilisé pour l'encapsulation. Toutefois, l'encapsulation selon l'invention ne se limite pas à cet hydrogel et d'autres matières d'encapsulation pourraient être choisie, comme le chitosan, les carraghénanes, les gels d'agarose, les polyéthylènes glycols (PEG), à titre non limitatif, à condition d'adapter l'unité d'encapsulation au type de gélification que requiert le polymère choisi.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.
De préférence, avant chaque encapsulation, l'on transfère les amas triés d'un milieu de culture les contenant vers la phase d'encapsulation destinée à les contenir, pour minimiser les pertes de charge lors du tri.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.
Egalement à titre préférentiel, le procédé selon l'invention comprend en outre un couplage fluidique entre le tri et l'encapsulation ayant pour effet de maintenir un régime fluidique laminaire dans les microcanaux correspondants, ce couplage faisant communiquer directement ou bien sélectivement lesdits amas triés avec la phase d'encapsulation.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.
Comme indiqué précédemment, on peut réaliser ce couplage au moyen de microcanaux intermédiaires de dimensions et de géométrie adaptées pour le maintien du régime laminaire lors du tri et de l'encapsulation.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.
En variante, on réalise préférentiellement ce couplage en réglant la concentration de chaque catégorie d'amas triés dans un microréservoir tampon de stockage des amas communiquant avec l'un desdits microcanaux de tri et relié sélectivement par ladite vanne fluidique à un microcanal de sortie véhiculant les amas triés et concentrés, l'ouverture et la fermeture de cette vanne abaissant et élevant respectivement la concentration des amas triés dans le microréservoir en fonction du nombre de capsules en cours de formation, pour minimiser la formation de capsules vides. Ce microréservoir est en outre avantageusement pourvu d'une pluralité de fins microcanaux transverses de sortie conçus pour évacuer la seule phase contenant ces amas sans ces derniers, lorsque la vanne est fermée.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.
Avantageusement, lesdits amas de cellules triés dans le procédé de l'invention sont des îlots de Langerhans qui sont encapsulés avec une taille de capsules variant de 70 µm à 200 µm pour les îlots triés selon une taille inférieure à 50 µm, avec une taille de capsules pouvant atteindre 650 µm pour les plus gros îlots triés selon une taille de 500 µm par exemple.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 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.
Une utilisation selon l'invention d'un système microfluidique tel que présenté ci-dessus consiste à trier soit des cellules, bactéries, organelles, liposomes, soit des amas de cellules, de préférence selon des catégories d'intérêt via des molécules d'adhésion dans le premier cas, ou bien selon des catégories de tailles dans le cas d'amas de cellules, puis à les encapsuler en continu et de manière automatisée pour chaque catégorie triée.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.
On notera en effet que l'invention n'est pas limitée au seul tri par taille puis à l'encapsulation d'amas de cellules, mais qu'elle vise d'une manière générale tout couplage d'une encapsulation à un tri préalable de cellules, de bactéries, d'organelles ou de liposomes au sein d'une population hétéroclite de ces particules très différentes, de façon à n'encapsuler que les cellules/bactéries/organelles/liposomes d'intérêt.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.
D'autres avantages, caractéristiques et détails de l'invention ressortiront du complément de description qui va suivre en référence à des dessins annexés, donnés uniquement à titre d'exemples et dans lesquels :
- 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 lafigure 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 lafigure 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 lafigure 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 lafigure 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 lafigure 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 lafigure 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 lafigure 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 lafigure 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 lafigure 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 desfigures 17a ou 17b , - les
figures 18 et19 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.
- 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 thefigure 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 thefigure 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 thefigure 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 thefigure 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 thefigure 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 thefigure 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 thefigure 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 thefigure 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 thefigure 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 theFigures 17a or 17b , - the
figures 18 and19 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.
Un système microfluidique 1 selon l'invention peut par exemple être réalisé comme suit, en référence aux
Un dépôt d'oxyde de silicium 4 (
Ce substrat 3 est ensuite gravé à la profondeur souhaitée des microcanaux par une gravure profonde 6 (
Les puces obtenues sont ensuite découpées et un capot de protection 2 en verre - ou en un autre matériau transparent pour permettre l'observation - est scellé, par exemple par scellement anodique ou scellement direct (
Avant montage des microcanaux ou capillaires (non illustrés), un traitement de surface du type silanisation hydrophobe peut aussi être effectué.Before mounting the microchannels or capillaries (not shown), a hydrophobic silanization surface treatment can also be performed.
Le protocole décrit ci-dessus est l'un des multiples protocoles de fabrication pouvant être suivis. Par ailleurs, on pourrait utiliser pour le substrat 3 un matériau autre que le silicium, par exemple un PDMS (polydiméthylsiloxane) ou bien un autre élastomère, par moulage sur un « master » (i.e. matrice) préalablement préparé par photolithographie par exemple. On notera que cette technique de fabrication est bien adaptée au cas ou le système microfluidique comporte un module de couplage entre l'unité de tri et celle d'encapsulation à vannes fluidiques, en référence aux
Le système microfluidique 101 selon l'exemple de l'invention illustré à la
Le principe de cette unité de tri 110 est illustré à la
La solution S permettant la focalisation des amas A à la paroi est injectée en un microcanal secondaire 116 communiquant avec le microcanal principal 115 par des embranchements 117 à 119, et cette solution S peut être la même que celle contenant les amas A injectés à l'entrée E de l'unité 110, étant par exemple un milieu de culture ou de l'alginate.The solution S for focusing the clusters A to the wall is injected into a
L'unité de tri 110 permet ainsi de trier des amas de cellules A, tels que des îlots de Langerhans, selon les quatre catégories suivantes :
- 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.
- 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.
En variante de la
Selon une autre variante de la
Comme visible à la
En revenant à la
La
- 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.
- 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 forencapsulation 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, amicrochannel 129 containing the oily phase.
Le module de gélification 135 illustré dans la variante de la
deux entrées 136et 137 comprenant :- * un microcanal
horizontal d'entrée 136 destiné à l'acheminement d'une phase huileuse contenant les amas de cellules At 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
- * un microcanal
deux sorties 138et 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 At 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.
- two
136 and 137 comprising:inputs - 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
- an inlet
- two
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:outputs - 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.
- an
Le module de gélification 145 illustré à la
Comme illustré à la
En variante de ces
Comme visible à cette
Puis les capsules sont mises en contact avec une solution de polycations de PLL (Poly-L-Lysine) dans un canal en forme de serpentin 227, qui permet d'ajuster le temps d'incubation des capsules dans cette solution de PLL. Les capsules sont par la suite rincées dans une solution de NaCl, pour éliminer le PLL non lié dans un second module de rinçage 228, et l'on élimine également ensuite la solution de NaCl de rinçage dans les microcanaux 229.The capsules are then placed in contact with a solution of PLL polycations (Poly-L-Lysine) in a
En dernière étape, les capsules sont recouvertes d'une couche externe d'alginate dans un module d'accrochage 230, pour l'obtention en sortie de l'unité 220 des capsules à trois couches Alginate-PLL-Alginate.In the last step, the capsules are covered with an outer layer of alginate in an
La
Les
En référence à ces deux
La fermeture de la vanne 32, 42 permet de stocker et surtout de concentrer les amas de manière que leur concentration dans la solution d'encapsulation soit suffisante pour limiter le nombre de capsules vides formées. Les microcanaux fins 34, 44 permettent de faire en sorte que la fermeture de la vanne 32, 42 ne modifie pas les lignes d'écoulement du fluide en amont dans l'étage de tri correspondant (la taille de ces microcanaux 34, 44 est telle que les amas ne peuvent pas y pénétrer et sont donc forcés de se concentrer dans le microréservoir 31, 41).Closing the
Plus précisément en référence à la
Plus précisément en référence à la
La position du gel 42a est déterminée par les pressions relatives des phases d'alginate, de Ca2+ et d'EDTA. Pour éviter que le microcanal 45 véhiculant l'alginate ne se bouche, on peut introduire une faible quantité d'EDTA en même temps que cet alginate.The position of the
Une fois l'étape de concentration des amas terminée et le gel d'alginate 42a dissous, la pression de circulation de l'EDTA (injecté dans deux microcanaux différents 46 et 47 opposés par rapport au microcanal de sortie 43) et la pression de circulation des ions Ca2+ (injectés dans un microcanal 48 adjacent à un microcanal 49 véhiculant le milieu de culture) peuvent être quasiment nulles: seuls l'alginate et ce milieu de culture, complètement inoffensifs pour la viabilité des amas, circulent alors dans la chambre 43. Cette dernière est en outre pourvue d'une sortie 50 pour l'acheminement des amas triés et concentrés vers la sous-unité d'encapsulation 121 à 124 correspondante, et d'une sortie 51 équipée de fins microcanaux de filtrage 51a pour l'évacuation des seuls ions Ca2+.Once the cluster concentration step is complete and the
On notera que le principal avantage de ce type de vanne 42 est qu'il n'y a aucune complication d'ordre technologique pour l'incorporer au microsystème selon l'invention.It will be noted that the main advantage of this type of
La
Plus précisément, cette unité d'encapsulation 320 comporte :
- 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 premièreentrée 321, - deux sorties opposées 323
et 324 pour la phase aqueuse introduite par la premièreentrée 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 premièreentrée 321.
- 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), thisinlet 321 defining ahorizontal microchannel 321a, - a
second inlet 322 of an immiscible phase with this aqueous phase (eg an oil, undecanol, "FC"), thisinlet 322 being provided opposite and below thefirst inlet 321, - two
323 and 324 for the aqueous phase introduced by theopposite outlets first inlet 321, which are provided below the latter but above thesecond inlet 322 and which are interconnected by two lateral microchannels 323a and 324a (horizontal) communicating with avertical microchannel 325 extending at right angles to themicrochannel 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 thesecond inlet 322 of this immiscible phase, forming therewith alower microchannel 327 encapsulation which communicates with thevertical microchannel 325 so as to receive by gravity clusters from thefirst input 321.
On notera que cette unité d'encapsulation 320, qui est formée en trois dimensions (en ce sens que les entrées et sorties microfluidiques 321, 322, 323, 324 et 326 ne sont pas situées dans un même plan), est apte à former les capsules C non seulement par la surpression locale précitée résultant de l'obstruction des deux microcanaux latéraux 323a et 324a, mais aussi par la force de sédimentation des amas de cellules due à la gravité.It will be noted that this
En conclusion et comme l'illustre à titre d'exemple la
- 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.
- 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.
De cette manière, on comprend que le procédé selon l'invention permet d'adapter au plus près la taille des capsules formées, suite au tri des amas de cellules, à la taille des diverses catégories d'amas triés. Il en résulte avantageusement :
- 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.
- 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.
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CN102530833B (en) * | 2011-12-02 | 2014-10-29 | 江苏大学 | Closed-type microfluidic channel etching method and automatic etching device |
US10946380B2 (en) | 2018-01-19 | 2021-03-16 | International Business Machines Corporation | Microfluidic chips for particle purification and fractionation |
US11458474B2 (en) | 2018-01-19 | 2022-10-04 | International Business Machines Corporation | Microfluidic chips with one or more vias |
US11566982B2 (en) | 2018-01-19 | 2023-01-31 | International Business Machines Corporation | Microscale and mesoscale condenser devices |
US11754476B2 (en) | 2018-01-19 | 2023-09-12 | International Business Machines Corporation | Microscale and mesoscale condenser devices |
US11872560B2 (en) | 2018-01-19 | 2024-01-16 | International Business Machines Corporation | Microfluidic chips for particle purification and fractionation |
CN109943475A (en) * | 2019-04-12 | 2019-06-28 | 广西医科大学第一附属医院 | The micro-fluidic sorting chip of one kind and its separation system |
Also Published As
Publication number | Publication date |
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EP2119503A3 (en) | 2012-02-22 |
FR2931141A1 (en) | 2009-11-20 |
JP2009273461A (en) | 2009-11-26 |
JP5414347B2 (en) | 2014-02-12 |
US8263023B2 (en) | 2012-09-11 |
FR2931141B1 (en) | 2011-07-01 |
EP2119503B1 (en) | 2015-04-01 |
US20090286300A1 (en) | 2009-11-19 |
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