EP2119503B1 - Système microfluidique et procédé pour le tri d'amas de cellules et pour leur encapsulation en continu suite à leur tri - Google Patents

Système microfluidique et procédé pour le tri d'amas de cellules et pour leur encapsulation en continu suite à leur tri Download PDF

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EP2119503B1
EP2119503B1 EP09290311.1A EP09290311A EP2119503B1 EP 2119503 B1 EP2119503 B1 EP 2119503B1 EP 09290311 A EP09290311 A EP 09290311A EP 2119503 B1 EP2119503 B1 EP 2119503B1
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Prior art keywords
sorting
clusters
encapsulation
sorted
capsules
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English (en)
French (fr)
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EP2119503A3 (fr
EP2119503A2 (fr
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Sophie Le Vot
Jean Berthier
Florence Rivera
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Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B01F33/301—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions
    • B01F33/3011—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/40—Mixing liquids with liquids; Emulsifying
    • B01F23/41—Emulsifying
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/40—Mixing liquids with liquids; Emulsifying
    • B01F23/41—Emulsifying
    • B01F23/414—Emulsifying characterised by the internal structure of the emulsion
    • B01F23/4145—Emulsions of oils, e.g. fuel, and water
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0688—Valves, specific forms thereof surface tension valves, capillary stop, capillary break
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • 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
    • Y10T436/11—Automated chemical analysis
    • 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
    • Y10T436/11—Automated chemical analysis
    • Y10T436/117497—Automated chemical analysis with a continuously flowing sample or carrier stream
    • 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
    • Y10T436/11—Automated chemical analysis
    • Y10T436/117497—Automated chemical analysis with a continuously flowing sample or carrier stream
    • Y10T436/118339—Automated chemical analysis with a continuously flowing sample or carrier stream with formation of a segmented stream
    • 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
    • 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 the 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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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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FR2931141B1 (fr) 2011-07-01
EP2119503A3 (fr) 2012-02-22
US20090286300A1 (en) 2009-11-19
FR2931141A1 (fr) 2009-11-20
JP2009273461A (ja) 2009-11-26
US8263023B2 (en) 2012-09-11
EP2119503A2 (fr) 2009-11-18

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