Classifications

• • 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
  • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
  • B01F33/30—Micromixers
  • B01F33/302—Micromixers the materials to be mixed flowing in the form of droplets
  • B01F33/3021—Micromixers the materials to be mixed flowing in the form of droplets the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• • 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
  • B01F33/3035—Micromixers using surface tension to mix, move or hold the fluids
• • 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
  • B01L3/502769—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 multiphase flow arrangements
  • B01L3/502784—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 multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0668—Trapping microscopic beads
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
• • 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
  • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
• • 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

Definitions

• the present invention relates to a microfluidic process for handling several microdroplets in at least one capillary trap of a microfluidic system.
• the invention also relates to a microfluidic device for implementing such a method.
• Such traps do not allow precise manipulation and / or control of the trapped microdroplets, in particular to adapt the traps to microdrops of different sizes, nor trapping the microdroplets in a predefined manner spatially.
• the application WO 2016/059302 describes a method of handling microdroplets in a microfluidic system comprising the step of trapping the microdroplets in a capillary trap and at least partially gel microdrops or their environment.
• the capillary trap can receive several microdrops in its depth, which is greater than the diameter of the trapped microdroplets.
• the handling of microdrops, in particular those trapped, in depth is limited.
• a microfluidic circuit is disclosed therein, which can return microdrops, one by one, into cavities serving as capillary traps.
• Two microdroplets of different volumes can be brought into contact and received in a cavity in the form of 8.
• the dimensions of the two trapping zones, each trapping area corresponding to a loop of 8, correspond to the dimensions of the microdrops which must be housed therein. .
• the application US 2010/0190263 describes a droplet actuator having recessed regions separating two substrates. These substrates include electrodes for transporting the droplets to the recessed regions.
• the actuator targets the formation and retention of a gas bubble in these recessed regions.
• the trapping force can result from the shape and size of the trap, the depth of the micro-channel, the size of the microdrops, the physical and physicochemical properties of the fluids present, such as the viscosity, the tension of area, ...
• the invention proposes, according to a first of its aspects, a method of handling at least a first microdrop and at least a second microdrop in a microfluidic system comprising a capillary trap having a first trapping zone and a second trapping area, the method comprising the steps of:
• first and second trapping zones being arranged so that the first and the second microdrop are in contact with each other
• the first and second trapping zones being shaped so that the trapping forces brought to one of said microdroplets are different.
• microfluidic system refers to a system involving the transport of at least one product and which comprises, on at least one of its portions, a section of which at least one dimension measured in a straight line from an edge to an edge opposite is less than one millimeter.
• microdrop it is understood a drop having a volume less than or equal to 1 ⁇ , better than or equal to 10 ni.
• the microdrop may be liquid, gaseous or solid.
• capillary trap is meant a spatial zone of the microfluidic system allowing the temporary or permanent immobilization of one or more microdrops circulating in the microfluidic system.
• the capillary trap may be formed by one or more reliefs, in particular recessed reliefs, and / or by one or more local modifications of the surface in contact with the microdroplets, in particular one or more local modifications of the affinity of the surface with at least a part of the contents of the microdrop.
• the trapping force of a trapping area depends in particular on its shape, its surfaces in contact with the microdrop and / or properties, including dimensions, trapping microdroplets.
• the capillary trap has two zones having different trapping forces brought back to one of the microdroplets makes it possible to have both a selectivity of the trapped microdroplets and a spatial selectivity, in particular to prevent the first microdrop from occupying the second trapping zone thus preventing the second microdrop from getting trapped in this second trapping area. This is particularly interesting when a plurality of first and second microdroplets are introduced into the microfluidic system.
• first and second microdroplets are in contact allows them to interact or coalesce.
• the first microdrop is trapped in the first trapping area with a trapping force which is greater than that which the second trapping area would exert on the first microdrop.
• the first microdrop is preferably trapped by the first trapping area.
• the second microdrop is trapped in the second trapping area with a trapping force that is smaller than that the first trapping area would exert on the second microdrop.
• the first and second microdroplets are preferably introduced and trapped in the microfluidic system sequentially.
• the first microdrop is trapped by the first trapping area in the microfluidic system before the second microdrop is trapped by the second trapping area in the microfluidic system.
• the second microdrop when introduced, it can not occupy the first trapping area because it is already occupied by the first microdrop.
• a driving force greater than the trapping force of the second trapping area and less than or equal to the trapping force of the first trapping area trapping can be exerted in step (i) on the first microdrop.
• the shape of the second trapping area is chosen so that the trapping force of the second area is less than the driving force.
• the microdrop is subjected to hydrodynamic forces due to its training that oppose its capture by the second trapping zone.
• the drag force exerted by the fluid that carries the microdroplets may depend on the size and the instantaneous shape of the microdrops, the physical and physicochemical properties of the fluids (viscosity, surface tension, etc.) and the speed of the microdroplets. flow.
• the first drop is then trapped only in the first trapping areas.
• the driving force can be exerted at least partially by
• the microdroplets may be driven in displacement in the microfluidic system by an oriented flow of a fluid, in which the microdrops are preferably immiscible, the fluid flow being such that, especially having a flow rate and a orientation such that the first microdrop is only trapped by the first trapping area.
• the force that the moving fluid exerts on the first drop prevents it from trapping in the second trapping area;
• the first microdrop being driven along a slope of the microfluidic system by its own weight.
• the first microdrop is or is not retained the first trapping area;
• the microfluidic system may comprise reliefs that cause the first microdrop in displacement, including a groove widening towards the capillary trap.
• the second microdrop is subjected to a driving force as described in relation to the first microdrop, less than or equal to the trapping force of the second trapping area.
• the driving force on the second microdrop is exerted by an oriented fluid flow
• the force exerted by the fluid flow on the first microdrop trapped in the first trapping zone is preferably insufficient to extract the first microdrop from the first trapping area.
• the latter can be oriented so that the second microdrop can be trapped only in the second trapping zones having a particular orientation relative to the orientation of the flow, in particular disposed upstream of the first trapping area with respect to the direction of the fluid flow.
• microdroplets being conveyed by a flow of fluid to a plurality of trapping zones, they are preferably brought into these randomly and naturally occupy the most advantageous place, from an energy point of view, the area of trapping. They lodge themselves in the trapping areas. This random placement of the microdroplets makes it possible to have a large number of microgoutts trapped simultaneously, and increases the screening capacity.
• the method comprises the following steps:
• the trapping force F1 exerted by the first zone on the first microdrop being greater than the force Ft1 of hydrodynamic drag exerted by the flow (ie the oriented flow) of fluid that carries the microdroplets) on the first microdrop, so that the latter remains trapped in the first zone, the drag force Ft1 being between Fl and F2, F2 denoting the trapping force exerted on the first microdrop by the second trapping area of the capillary trap,
• the hydrodynamic drag force Ft2 exerted on the second microdrop by the flow during loading of the second microdrop in the second zone being between F2 and F3, with F3 preferably lower than F1, F3 being the trapping force exerted by the second zone of the capillary trap on the second microdrop.
• the trapping force exerted by the first trapping area on the first microdrop is different from the trapping force it would exert on the second microdrop and the trapping force exerted by the second trapping area on the second microdrop is different from the trapping force it would exert on the first microdrop.
• the trapping force also depends on the shape of the microdrop trapping in relation to the shape of the trapping area. This facilitates the sequential trapping of the first and second microdrops.
• the first trapping area exerts on the first microdrop a trapping force greater than that which it would exert on the second microdrop. This prevents the second microdrop does not dislodge and takes the place of the first microdrop.
• the first and the second microdrop may be different, in particular of different sizes, in particular the first microdrop being larger in size and / or larger in volume than the second microdrop, and / or of different contents. This ensures that the first trapping area exerts on the first microdrop a trapping force greater than that which it would exert on the second microdrop; therefore, there will be only one microdrop in the first trapping area.
• the first trapping area may trap one or more first microdrops.
• the second trapping area may trap one or more second microdrops.
• the first and second microdroplets are different by at least one of their properties, in particular their viscosity and / or their interfacial tension and / or their affinity with a particular coating of at least one of their properties. one of the trap areas.
• the first trapping area traps only a first microdrop and / or the second trapping area traps only a second microdrop.
• the first trapping area traps only a first microdrop and / or the second trapping area traps only a second microdrop.
• the largest dimension of the first microdrop when it is trapped in view from above may be greater than or equal to the largest dimension of the first trapping area in top view, and / or
• the largest dimension of the second microdrop when it is trapped in view from above may be greater than or equal to the largest dimension of the second trapping area in top view, and / or the first microdrop, when trapped, fills at least 70%, better 80%, even better 90%, of the volume of the first trapping area, and / or
• the second microdrop when trapped, fills at least 70%, better 80%, still better 90%, of the volume of the second trapping zone, and / or
• the volume of the first microdrop, when trapped is greater than or equal to that of the first trapping area so that the first microdroplet extends partially outside the first trapping area, and / or
• the volume of the second microdrop, when trapped, is greater than or equal to that of the second trapping area so that the second microdroplet extends partially outside the second trapping area.
• One of the first (s) or second (s) microdrops can be a microbubble air.
• the capillary trap may comprise a plurality of second trapping zones, the step (ii) of trapping a second microdrop by a second trapping zone, the first and the second trapping zones being arranged in such a way that each second microdrop is in position. contact with at least one of the first or second microdroplets.
• the capillary trap may comprise a plurality of first trapping zones, the step (i) of trapping a first microdrop by a first trapping zone, the first and the second trapping zones being arranged in such a way that each first microdrop or in contact with at least one of the second or second microdrops or first microdrops.
• each second microdrop is connected to the or each first microdrop.
• each second microdrop is either directly in contact with said first microdrop, or in contact with another second microdrop or a string of second and / or first microdrops itself in contact with said first microdrop.
• string of microdrops is meant a plurality of microdroplets forming a straight or curved line in contact with each other.
• the second microdroplets may all be in contact with at least a first microgout trapped in said capillary trap.
• At least two second trapping zones may be shaped so that their trapping forces brought to one of said second microdrops are different.
• the second microdroplets trapped by at least two second trapping zones may be different by at least one of their properties, in particular their larger size.
• all the second trapping areas of the capillary trap are identical.
• the microfluidic system may comprise a plurality of capillary traps each comprising a first trapping area and a second trapping area, the step (i) of trapping a first microdrop in the first trapping area of each capillary trap, step (ii) of trapping a second microdrop in the second trapping area of each capillary trap, wherein the first and second trap areas of each capillary trap of the plurality of traps are arranged such that the first and second microgout trapped in said capillary trap are in contact with each other in the latter.
• Each capillary trap may comprise one or more of the features described above.
• All traps of the microfluidic system may each have a first trapping area and a second trapping area arranged so that the first and second trapping microdrop trapped in said trapping capillary are in contact with each other therein.
• only a portion of the capillary traps of the microfluidic system each comprise a first trapping area and a second trapping area arranged so that the first and second microdrop trapped in said capillary trap are in contact with each other. other in the latter.
• the method may comprise the step of trapping a microbubble of gas, in particular air, in one of the first or second trapping zones. This makes the trapping area in question inactive. Indeed, because of the presence of microbubble gas, the first or second microdrops can not be trapped in the trapping area concerned.
• Step (ii) may comprise the substeps ( ⁇ ') of trapping, under the effect of a first oriented fluid flow, a second microdrop in one or a part of the second trapping zones and (ii " ) trapping, under the effect of a second oriented fluid flow, a second microdrop in another or another part of the second trapping areas, the first and the second fluid stream being of different orientation.
• the second microdrops of the steps ( ⁇ ') and (ii ") can be different, in particular by at least one of their properties and / or their content,
• the second trapping zones of the steps (ii') and (ii") can to be identical.
• microdrops it is possible, by choosing the orientation of the fluid flow, to trap microdrops selectively in one of the two trapping zones, which allows a predefined spatial positioning of the microdroplets in contact with each other. It is then possible to bring a first microdrop into contact with different second microdrops in a controlled manner, particularly in the context of combinatorial chemistry. It is also possible, in the context of microdrops comprising gels to spatially control the arrangement of microdrops of gels in the capillary trap so as to obtain after melting a microdroplet of controlled shape and composition.
• the method may comprise step (iii) of fusing with the first microdrop the or each of the second microdrop trapped in the or each of the second trapping zones. Such coalescence makes it possible in particular to mix the contents of the two microdrops.
• Such a coalescence can be selective, that is to say that one can choose the second or microdroplets in contact with the first microdrop that is to be fused with the latter, in particular by the use of a laser infra-red as described, for example, in E. Fradet, P. Abbyad, MH Vos, and CN Baroud, Parallel measurements of reaction kinetics using ultralow-volumes. "Lab Chip, Vol 13, No. 22, pp. 4326-30, Oct. 2013, whose contents are incorporated by reference, with addressable electrodes disposed at the capillary trap or mechanical waves.
• the coalescence of the microdroplets is non-selective, ie all of the second microdroplets of the capillary trap merge simultaneously with the first microdrop, in particular by adding a product that promotes this coalescence in the microdrop.
• capillary trap environment or the application of an external physical stimulus such as mechanical waves, pressure waves, a temperature change or an electric field.
• step (iii) is to evacuate the at least one second microgout trapped in the second trapping area out of the capillary trap.
• Step (iii ') may consist in applying an oriented fluid flow, configured to exert on a second or more second microdrops a driving force greater than the trapping force of the second trapping area, the fluid flow being configured to exert on the first microdrop or drops a driving force less than or equal to the trapping force of the first trapping area, so that the first microdrop or drops remain trapped in the first trapping area.
• one or more of the second microdroplets of the second trapping zones can be evacuated, the capillary trap being configured so that, because of its orientation, the fluid flow exerts different driving forces on the second zones of the trapping zone.
• the method preferably comprising step (iv) of changing the orientation of the fluid stream so as to evacuate at least one or more of the second microdroplets from at least one other trapping area. This allows a selective release of the second microdrops.
• a first microdrop and one or more second microdroplets can be brought into contact with each other for a sufficient time sufficient so that, particularly because of the interactions between the first microdrop and the second microdrop or microdroplets, the second or second microdroplets microdrops undergo a change, for example a change of content, and then released for analysis.
• This can also make it possible to change the second microdroplets by other second microdrops in the case of an error in the protocol before the coalescence of microdrops.
• the method may comprise, after step (iii) or (iii '), step (v) of trapping a third microdrop in the second trapping zone or zones having no second microdrop, so that the first and third microgoutts are in contact with each other.
• the third microdrop may be the same or different of the second microdrop.
• the third microdrop may be fused with the microdrop trapped in the first trapping area or released as previously described for the second microdrop.
• Step (vi) can be repeated several times. This allows for example:
• the method may comprise the step (vi) of evacuating all the microdroplets present in the capillary trap from the capillary trap, in particular by means of a fluid flow exerting a driving force greater than the trapping forces. exercising on microdrops. Such a step can make it possible to release the microdrops to analyze them.
• the method may include the step of taking a measurement of the state of the microfluidic system. This measurement can be performed before and / or after melting and / or releasing the drops.
• the final microduct (s) obtained may comprise a means for identifying their contents, in particular a marking by the presence of beads or particles in number, by the presence of various colors or shapes and / or by a colorimetric signal. or fluorescence proportional to an initial concentration of a compound included in one of the first and second microdrops.
• the method may include an additional step
• the observation or measurement step may make it possible to determine the contents of each microdrop before and / or after melting and for example to determine the changes that have occurred following the melting.
• the observation step is, for example, particularly useful in the context of the use of a library of different microdrops for mapping the various microdrops before melting.
• the invention also relates to a micro-fluidic device for trapping microdroplets, in particular for implementing the method according to any one of the preceding claims, comprising a capillary trap having a first trapping area and a second trapping area arranged. so that a first microgout trapped in the first trapping area and a second microgout trapped in the second trapping area are in contact with each other in the capillary trap, the first and the second trapping area being shaped so that the trapping forces brought back to one of said microdroplets are different.
• the capillary trap has two zones having different trapping forces brought back to one of the microdroplets makes it possible to have both a selectivity of the trapped microdroplets and a trapping of the microdroplets in a spatially predefined manner, in particular to avoid that the first micro-droplet occupies the second trapping zone thus preventing the second microdrop from getting trapped in the latter.
• first and second microdroplets are in contact makes it possible for them to interact, or to be able to easily merge them. Trapping areas
• the first and second trapping areas are preferably cavities.
• the use of trapping zones in the form of a cavity facilitates the handling of the microdrops and in particular their entrapment and / or their release.
• the first and second trapping areas may be disjoint.
• first and second trapping areas are joined.
• the capillary trap is devoid of symmetry of revolution, seen from above. This anisotropy makes it possible to trap the microdrops in a spatially predefined manner.
• the first trapping area and the second trapping area are arranged next to one another, in view from above.
• the first and second trapping areas are preferably different by at least one of their dimensions.
• the first and second trapping areas are of different heights, the first trapping area being in particular higher than the second trapping area or the first and second trapping areas are of different shapes, seen from above, the first trapping area having in particular a larger section than the second trapping area.
• the difference in entrapment force is then at least partially related to the size, in particular the height or the section in view from above, of the trapping zones.
• height of the trapping area is understood, in cross-section, the average height of the trapping area of the microfluidic system.
• the second trapping area may expand in at least one direction towards the first trapping area. This guides the second microdrop in the direction of the first microdrop to keep in contact with the latter. Indeed, in order to minimize its surface energy, the second microdrop tends to move along the second trapping area to the larger area.
• the second trapping area may widen towards the first trapping area, seen from above.
• the angle of divergence a is such that the second microdrop is always in contact with the two opposite walls defining it.
• the second trapping zone may widen with an angle of divergence a non-zero, in particular between 10 ° and 120 °.
• the second trapping area may have a substantially triangular or truncated triangular shape.
• the second trapping area may have increasing height towards the first trapping area.
• the height of the second trapping area is less than or equal to the largest dimension of the first trapping area, more preferably at half the largest dimension of the first trapping area.
• the fact that the height of the second trapping area is limited makes it possible to prevent the fluid flow lines from being disturbed by the second trapping area to the point of preventing the second microdrop from being trapped.
• the height of the first trapping zone may be such that the volume of the latter is greater than or equal to the volume of the first microdrop. This makes it possible to have a first trapping zone having a high trapping force, in which the first microdrop is slightly deformed, in particular having a concave lower interface, which can facilitate, after sedimentation, the contacting of encapsulated elements. to form a cluster, for example cells to form a spheroid.
• the capillary trap may comprise a plurality of second trapping zones arranged such that each trapped second microgout is in contact with at least one of the first or second microdrops trapped in the capillary trap.
• the capillary trap may comprise a plurality of first trapping zones arranged such that each first trapped microgout is in contact with at least one of the second or second microdroplets or first microdroplets trapped in the capillary trap.
• the first (s) and second (s) trap areas are arranged so that each second microdrop is connected to the or each first microdrop.
• the first and second trapping zones may be arranged such that the second microdroplets are all in contact with at least a first microdrop trapped in said capillary trap.
• At least two second or first trapping zones may be shaped so that their trapping forces brought to one of said second microdrops are different.
• the second microgout trapped by at least two second trapping areas may be different by at least one of their properties, including their largest dimension.
• all the second trapping areas of the capillary trap are identical.
• the device comprises a plurality of capillary traps each comprising a first trapping area and a second trapping area, preferably arranged so that the second microdrop trapped in the second trapping area of the capillary trap is in contact with the trapping zone. first microgout trapped in the first trapping area of said capillary trap.
• Each capillary trap may comprise one or more of the features described above.
• All capillary traps of the device may each comprise at least a first trapping area and at least a second trapping area.
• a portion of the capillary traps each comprise at least one first trapping zone and at least one second trapping zone and part of the traps having only one trapping zone that can trap only a single first microdrop .
• Such capillary traps with a single trapping area can serve as a control during an experiment.
• the device may comprise at least 10 capillary traps per square centimeter, more preferably at least 100 capillary traps per square centimeter.
• a large number of capillary traps makes it possible in particular to make combinatorial chemistry, to carry out a drug screening, to study the crystallization of proteins, to carry out a titration of a chemical species, or to customize a treatment, particularly in the case of cancer treatment.
• At least two capillary traps can be different.
• the device comprises a first capillary trap having n second trapping zones and a second capillary trap having p second trapping zones, n being different from p.
• Such capillary traps make it possible to have after coalescence of the second microdroplets with the first microdroplets microdroplets trapped in the first trapping areas having different concentrations and / or drop sizes.
• the microfluidic system may have more than two capillary traps having different amounts of second trapping zones in order to achieve several concentration and / or size of microdroplets, in particular a concentration gradient. and / or microdrop sizes.
• microdroplets obtained can form a panel of microdroplets useful in the field of combinatorial chemistry, to study the crystallization of proteins, to perform a titration of a chemical species or to customize a treatment, particularly in the case of cancer.
• the capillary traps are all identical.
• the device may comprise a channel having a trapping chamber, the cap or traps being in the trapping chamber.
• the invention also provides, in a second aspect, a method of handling a plurality of first microdroplets and a plurality of second microdrops in a microfluidic system having a channel having a trapping chamber having a plurality of capillary traps. distributed in at least two different directions, each capillary trap having a first trapping area and a second trapping area, the method comprising the steps of:
• the first and second trapping areas of the same capillary trap being arranged so that the first and the second microdrop are in contact with each other in the capillary trap, the capillary traps each having an anisotropic shape.
• capillary traps are anisotropic makes it possible to have a predefined spatial positioning of the microdroplets once they are trapped by the trapping zones.
• the first and the second trapping area of each capillary trap are shaped so that the trapping forces brought to one of said microdroplets are different.
• One or more of the features described above in connection with the method or device according to the preceding aspects of the invention can be applied to the method according to this aspect of the invention.
• the method can be implemented using a microfluidic microdrop trapping system comprising a channel having a trapping chamber comprising a plurality of capillary traps distributed in at least two different directions, each capillary trap having a first zone of trapping and a second trapping area arranged so that a first microgout trapped in the first trapping area and a second trapping microgout trapped in the second trapping area of the same trapping trap are in contact with each other, capillary traps each having an anisotropic form.
• the first and the second trapping area of each capillary trap are shaped so that the trapping forces brought to one of said microdroplets are different.
• the invention further provides, according to a third aspect, a method of handling at least a first microdrop and at least a second microdrop in a microfluidic system comprising a capillary trap having a first trapping area and a second area trapping zone, the second trapping area widening in at least one dimension towards the first trapping area, the method comprising the steps of:
• the second trapping area widens in at least one dimension towards the first trapping area makes it possible to guide the second microdrop to the first microdrop during its trapping and to keep it in contact with the first microdrop. Indeed, in order to minimize his Surface energy, the second microdrop tends to move along the second trapping area to the larger area.
• the second trapping zone widens, preferably, in plan view towards the first trapping area.
• the second trapping area may widen with a divergence angle ⁇ between 10 ° and 120 °.
• the second trapping area may have increasing height towards the first trapping area.
• the first and second trapping zones are shaped so that the trapping forces brought to one of said microdroplets are different.
• the method can be implemented using a microfluidic microdrop trapping system having a capillary trap having a first trapping area and a second trapping area arranged so that a first microdrop trapped in the first trapping area. trapping and a second microgout trapped in the second trapping area of the same capillary trap are in contact with each other, the second trapping area widening in at least one dimension towards the first trapping area .
• the first and second trapping zones are shaped so that the trapping forces brought to one of said microdroplets are different.
• the invention further provides, according to a fourth aspect, a method of cell assembly of at least a first microdrop containing first cells and at least a second microdrop containing second cells, in a microfluidic system comprising a trap capillary having a first trapping area and a second trapping area, said method comprising the steps of:
• Such a method can make it possible to create in vitro microtissues with a controlled architecture to mimic very faithfully the conditions encountered in vivo.
• the different cell types are often arranged in tissue according to a specific architecture that is important to reproduce at best to recreate a function at an organ.
• This three-dimensional architecture with controlled architecture can be used for transplantation on a patient. For example, it is possible to culture insulin-producing alpha cells, glucagon producers, and beta cells to create Langerhans islands that can be transplanted into a patient's pancreas to heal. his diabetes. Similarly, hepatocytes and stellate cells may be associated in liver transplantation.
• Step (ii) can be performed after aggregation of the first cells, in particular after formation of a first spheroid formed by adhesion of the first cells together. If the first microdrop containing the first spheroid is liquid, the second cells will, after fusion of the two microdrops, mix with the contents of the first microdrop and then sediment to reach directly the first spheroid. If step (iii) takes place before the second cells have had time to form a second spheroid, they will be deposited after sedimentation on the surface of the first spheroid initially in the first microdrop.
• Step (iii) can be performed after aggregation of the second cells, in particular after formation of a second spheroid formed by adhesion of the second cells to each other.
• the first and second spheroids can be fused together.
• the architecture of the microtissues obtained therefore depends on the experimental conditions.
• the process may comprise an additional step of gelation of the first microdrops, such a step taking place before step (iii) and preferably before step (ii).
• This makes it possible to compartmentalize the cells. Indeed, if the first microdrops, which contain the spheroids are gelled before the arrival of the second microdrops, the second contained cells can no longer, after coalescence, come into direct contact with the first spheroid, for example mammalian cells can not pass through an agarose matrix at 0.9% by weight. The first and second cells can then communicate together only paracrine.
• the first and second cells may be of different cell types.
• the first and second trapping zones are shaped so that the trapping forces brought to one of said microdroplets are different.
• the method can be implemented using one of the microfluidic systems according to the preceding aspects.
• the subject of the invention is also a method of cell culture in a microfluidic system of at least a first microdrop containing a cell culture and at least a second microdrop containing a culture medium, the microfluidic system. having a capillary trap having a first trapping area and a second trapping area, the culture method comprising the steps of:
• (iii) fusing the first microdrop with the second microdrop to renew the culture medium of the cell culture operated in the first microdrop.
• the sequential injection of the culture medium can be used to renew the latter several times, for example to allow the culture of the cell or cells in the first microdrop.
• the second microdrop may include an active test to model the intermittent nature of taking a drug.
• an active test to model the intermittent nature of taking a drug.
• a drop containing a spheroid of mammalian cells can be fused every 6 hours with a microdrop containing an active agent to be tested, in particular a drug.
• the method may comprise, after step (iii), step (iv) of repeating steps (ii) and (iii) to further renew the culture medium of the cell culture operated in the first microdrop.
• the first and second trapping zones are shaped so that the trapping forces brought to one of said microdroplets are different.
• the method can be implemented using one of the microfluidic systems according to the preceding aspects.
• Another object of the invention is, according to a sixth aspect, a method of forming multilayer gelled microdroplets of at least a first microdrop of a first gellable medium and at least one second microdrop of a second gellable medium in liquid form in a microfluidic system comprising a capillary trap having a first trapping area and a second trapping area, said method comprising the steps of:
• the method may comprise a step (v) occurring before or after step (iv) and comprising gelation of the second gellable medium.
• step (v) takes place after step (iv)
• the gelation makes it possible to form an outer layer of the second gel on the first microdrop.
• This makes it possible to form complex gel microdrops having radially variable mechanical and / or chemical properties.
• Such gel microdrops could be used with stem cells whose differentiation is, in particular, controlled by the rigidity of the gel.
• Microdroplets of gels with different hydrogel layers containing different cell types can also make it possible to model the different layers of the skin in the context of cosmetic tests.
• a microdroplet having a collagen core and an outer layer of agarose with sufficiently small pores can be used to create a spheroid of neurons from which only the axonal projections can be extracted through the pores of the outer layer.
• step (v) takes place before step (iv)
• the second gellable medium is gelled in the second trapping zone.
• the microdrops formed then retain the shape and arrangement of the first and second microdrops before melting.
• the arrangement, the shape and the number of the different trapping zones thus make it possible to directly control the shape of the final microdrop.
• Such microdrops can be used to model complex shapes.
• the controlled forms of the microdrops can also serve as an identifier of the latter.
• Step (v) can take place before or after step (iii) of trapping in the second trapping area.
• Step (ii) may take place before or after step (i) of trapping in the first trapping area.
• the method preferably comprises a step (vi) of repeating steps (iii) to (v).
• the first and second gellable media may be different.
• the first and second trapping zones are shaped so that the trapping forces brought to one of said microdroplets are different.
• One or more of the features described above in connection with the methods or devices according to the foregoing aspects of the invention can be applied to the method according to this aspect of the invention.
• the method can be implemented using one of the microfluidic systems according to the preceding aspects.
• the invention further relates, in a seventh aspect, to a method for encapsulating at least a first microdrop and at least a second microdrop in a microfluidic system comprising a capillary trap having a first trapping area and a second trapping area, one of the first microdrop and the second microdrop comprising a gelling medium and the other comprising a plurality of cells, said method comprising the steps of:
• This method makes it possible in particular to obtain spheroids encapsulated in biological hydrogels. Indeed, to be able to form spheroids in microdrops in a controlled manner, it must be possible to keep the contents of the liquid drop during the time of formation of the spheroid.
• Agarose lends itself very well to this protocol because it is a thermosensitive hydrogel. It remains liquid at 37 ° C and then solidifies after 30 min at 4 ° C and remains solidified after returning to 37 ° C. Only mammalian cells can not adhere to agarose and they can not digest it either. This matrix is therefore very different from the extracellular matrix found in the body.
• hydrogels such as, for example, type I collagen, fibronectin, Matrigel® or gelatin may be preferable to better mimic natural conditions. Only the control of their gelation is more difficult. For example, one can not keep liquid type I collagen for a long time with favorable conditions for culturing cells (low temperature or acidic pH). If one encapsulates cells in a drop of collagen that is made to gel quickly after trapping the cells, rather than adhere to each other and form a spheroid, cells will adhere to collagen and migrate individually along its fibers.
• This problem can be solved by the method above. Indeed, one can encapsulate cells in the first liquid microdrops within the first trapping area so as to form a spheroid. We can then bring second microdrops that will lodge in the second trapping area and contain one of the biological hydrogels mentioned above, especially in high concentration. Once these trapped second microdroplets are immediately fused first and second microdroplets in contact and the biological hydrogel, still liquid, will mix with the first microdrop that contains the spheroid. The gelation can then take place and will therefore encapsulate the spheroid in an extracellular matrix representative of the biological conditions encountered in vivo.
• Step (ii) can be carried out after aggregation of the first cells, in particular after formation of a spheroid formed by adhesion of the first cells to one another.
• the first and second trapping zones are shaped so that the trapping forces brought to one of said microdroplets are different.
• the method can be implemented using one of the microfluidic systems according to the preceding aspects.
• the subject of the invention is also a method of diluting a compound of interest in a microfluidic system comprising a first capillary trap comprising a first trapping zone and n second trapping zones and a second capillary trap. having a first trapping area and p trapping second areas, n being different from p, said method comprising the steps of:
• a spatial concentration gradient can be obtained after coalescence with the second microdroplets which may for example contain a diluent.
• Such a method can make it possible to obtain a panel of microdrops with different controlled concentrations, from microdrops of the same concentration. This may be of interest, for example, in forming a panel of microdrops of different concentrations for use in combinatorial chemistry, in the study of protein crystallization, in a subsequent method of titrating a chemical species, or in the personalization of treatment, especially in the case of cancer.
• the first and the second trapping area of each capillary trap are shaped so that the trapping forces brought to one of said microdroplets are different.
• the method can be implemented by the microfluidic microdrop dilution system comprising a first capillary trap having a first trapping area and n second trapping areas and a second capillary trap having a first trapping area and p trapping second areas, n being different from p, the first and second capillary traps being configured so that the second microdrop trapped in each of the second trapping zones are in contact with the first microdrop trapped in the corresponding first trapping area in the first and second capillary traps.
• the first and the second trapping area of each capillary trap are shaped so that the trapping forces brought to one of said microdroplets are different.
• the invention further provides, according to a ninth aspect, a method of screening a plurality of first microdroplets with a plurality of second microdroplets in a microfluidic system having a plurality of capillary traps, each capillary trap having a first trapping and a second trapping zone, the first microdroplets forming a first panel of identical or at least different microdroplets and the second microdroplets forming a second panel of microdroplets at least z are different, the method comprising the steps of:
• each capillary trap being arranged so that the first and second microgout trapped in said capillary trap are in contact with each other in the latter
• the fact that the microdroplets are static during the reaction facilitates the obtaining of kinetic data.
• the first panel of microdrops may include microdrops at least in different content, including their concentration in a first compound of interest.
• the second panel of microdrops may include microdrops at least different in content, including their concentration in a second compound of interest.
• the first or second microdrops can be obtained by the method according to the ninth aspect of the invention.
• the first and second compounds may be compounds that react together and whose initial concentrations are to be optimized. Thus we will be able to perform several reactions in parallel on a small volume to determine the initial compound concentrations giving the best results.
• the method comprises an additional step (iv) of observation or measurement, in particular by taking an image, by a colorimetric measurement, fluorescence, spectroscopic (UV, Raman) or temperature, before the step (iii).
• a step of observation or measurement in particular by taking an image, by a colorimetric measurement, fluorescence, spectroscopic (UV, Raman) or temperature, before the step (iii).
• a colorimetric measurement, fluorescence, spectroscopic (UV, Raman) or temperature before the step (iii).
• the method comprises an additional step (v) of observation or measurement, in particular by taking an image, by a colorimetric measurement, fluorescence, spectroscopic (UV, Raman) or temperature, after the step (iii).
• step (v) of observation or measurement in particular by taking an image, by a colorimetric measurement, fluorescence, spectroscopic (UV, Raman) or temperature, after the step (iii).
• the first panel of microdroplets comprises proteins, the first microdroplets being identical, and the second panel of microdroplets comprises at different concentrations a solution for the crystallization of proteins, including a saline solution.
• the process can then be used to study the crystallization of proteins as a function of the concentration of crystallization solution. Indeed, the optimal crystallization conditions vary from one protein to another.
• the first panel of microdroplets comprises a compound, the first microdroplets being identical and the second panel of microdroplets comprises at different concentrations a titrant species.
• This application can be particularly interesting in the case of the assay of expensive reagents or available in small quantities.
• the first panel of microdroplets comprises one or more cells and the second microdrops each comprise a drug to be screened at a determined concentration.
• the liver cells are cultured in the form of spheroids in the first microdrops and the supply in each of the second trapping zones of a second microdrop containing a drug with different concentrations, the aim of which is to evaluate the toxicity.
• the concentration that kills half of the cell population can be determined.
• This method can also make it possible to evaluate the interactions between different antibiotics. It is possible to create microdroplets forming a panel of microdrops having different antibiotic A and B concentrations and to fuse them with microdrops containing bacteria. The microdrops containing the bacteria can form a panel of microdrops of concentrations of different bacteria. This makes it possible to vary three different parameters in a single trapping chamber, namely the concentration of antibiotic A, the concentration of antibiotic B and the initial concentration of bacteria.
• microfluidics also allows the use of very small volumes, which can be very advantageous in the context of rare samples such as cells from a biopsy.
• the system can for example be used in the context of personalized medicine and cancer treatment. With this system it is possible to culture, for example in the form of spheroids in the first microdrops, tumor cells of a patient who has undergone a biopsy and to subject them to different active agents at multiple concentrations via the addition of the second microdrops. . After fusion of the cell-active microdrop couples, it is possible to determine which active will be the most effective, and in what concentration, for a particular patient, using only one chip and a minimal number of cells from the biopsy.
• the first and second trapping areas of the same capillary trap are shaped so that the trapping forces brought to one of said microdroplets are different.
• FIG. 1A illustrates in cross-section a capillary trap according to the invention
• FIG. 1B is a top view along I of the capillary trap of FIG.
• FIG. 2 diagrammatically shows a top view of the capillary trap of FIG. 1 after trapping two microdots
• FIGS. 3 and 4 are, in view from above, variations of capillary trap with microdroplets
• FIG. 5A represents in cross section a variant of a capillary trap
• FIG. 5B is a top view along V of the capillary trap of FIG.
• FIGS. 6 to 44 are variants, seen from above, of capillary traps with microdroplets
• FIG. 48 represents, in cross-section, a variant of a capillary trap
• FIG. 49 schematically shows, in plan view, a trapping chamber
• FIG. 50 is a diagrammatic representation, seen from above, of a trapping chamber
• FIG. 51 is a cross-sectional view of a capillary trap
• FIG. 52 is a schematic representation of a method according to the invention.
• FIG. 53 illustrates an alternative method for handling microdroplets in a capillary trap according to the invention
• FIGS. 54 to 59 illustrate variants of methods of handling microdroplets in a capillary trap according to the invention
• FIGS. 60 to 62 illustrate examples of implementation of the invention
• FIGS. 63 to 65 illustrate, in view from above, variants of capillary traps
• FIG. 66 illustrates the capture of microdroplets by an example of a capillary trap comprising two zones exerting different trapping forces on first and second microdroplets.
• the invention relates to a method of handling at least a first and a second microdrop in a microfluidic system.
• the microfiuidic system 5 comprises an upper wall 7 and a lower wall 8 forming between them a channel 9 for circulating the microdroplets and at least one capillary trap 12.
• the capillary trap 12 forms, in cross section of the microfiuidic system, a cavity in the lower wall 8 of constant height in which microdrops can be housed. It has, in view from above, a first trapping area 15 of circular shape on which is leaned a second trapping area 18 of triangular shape.
• the first and second trapping zones 15 and 18 exert different trapping forces on a given microdroplet, in particular because of their difference in shape.
• the first trapping area 15 exerts a trapping force larger than the second trapping area 18.
• first microdrop 20 When introducing a first microdrop 20 into the microfluidic system, the latter is trapped in the trapping zone having, for this microdrop, the greatest trapping force, here the first trapping area 15.
• the second microdrop 25 During the introduction of the second microdrop 25, the latter is trapped in the free trapping zone, here the second trapping zone 18, as illustrated in FIG.
• first microdrops 20 are shown in black and the second microdrops 25 are shown transparent without this representing a difference in particular content between the two microdrops.
• the first trapping area 15 has a diameter a substantially equal to the apparent diameter Di, seen from above, the first microdrop 20, once trapped in the first trapping area.
• the two trapped microgoutlets 20 and 25 are in contact with each other, in particular because of the small distance between the two trapping zones 15 and 18 relative to the diameters of the two microdroplets 20 and 25.
• the two microdroplets 20 and 25 are kept in contact because of the triangular shape of the second trapping area 18.
• the second microdrop 25, in contact with two opposite walls 27 and 28 of the second trapping area 18 moving away from each other. from one another towards the first trapping zone 15, is driven, by its natural tendency to always minimize its surface energy, to move in translation between the two opposite walls 27 and 28 towards the widening walls 27 and 28, ie towards the first trapping area 15 and therefore the first microdrop 20.
• the two walls 27 and 28 form between them an angle of divergence a substantially equal to 45 °.
• the first microdrop 20 has a diameter Di greater than that D 2 of the second microdrop 25.
• the second trapping zone 18 exerts on the second microdrop 25 a greater trapping force than it would exert on the first microdrop 20.
• the diameter of the second microdrop 25 better suits the shape and size of the second trapping zone 18 than that of the first microdrop 20. But it may be otherwise and the second microdrop 25 may be of the same diameter as the first microdrop 20.
• first and second microdrops 20 and 25 are different from each other by another of their properties, including their surface condition, viscosity or weight.
• the capillary trap 10 comprises two juxtaposed disjoint cavities respectively forming the first trapping area 15 of circular shape and the second trapping area 18 of triangular shape.
• the two trapping zones 15 and 18 of the capillary trap are sufficiently close so that the two microdroplets 20 and 25 trapped are in contact with each other.
• the distance e between the centers of gravity of the trapping areas 15 and 18 is smaller, as illustrated in FIG. 4, or equal, as illustrated in FIG. 3, to the sum SR of the radii of the two microdroplets.
• the first trapping area may be hexagonal in shape and the first trapping area 15 may have a height hi different from that h 2 of the second trapping zone 18, in particular hi is greater than h 2 .
• the first trapping area 15 then exerts a trapping force greater than the second trapping area 18.
• the second trapping area 18 is in the form of a long triangle having a low angle of divergence, here substantially equal to 10 °, making it possible to trap a string of second microdrops 25, here two seconds microdroplets 25a and 25b, in contact with each other. It can then be considered that the second trapping area 18 is in fact composed of two second trapping areas 18a and 18b each for trapping a second microdrop 25a and 25b.
• the second microdrop 25a is here connected to the first drop 20 via the second microdrop 25b.
• the trapping forces exerted by the second trapping areas 18a and 18b on the second microdrops 25a and 25b may be different depending on their positions.
• the second trapping area 18b exerts a stronger trapping force on the second microdrop 25b closest to the first trapping area 15. But it could be otherwise depending on the shape of the second trapping area 18.
• the two second trapping zones 18a and 18b can exert identical forces on the second trapped microdrops 25a and 25b.
• the capillary trap may have more than two second trapping zones configured to form a string of second trapped microdrops itself in contact with at least one of the second microdroplets forming it, with the first microdrop trapped in the first trapping area.
• capillary traps comprising a plurality of entrapment zones configured so that the trapped microdroplets are all interconnected directly or via the other microdroplets.
• the capillary trap 12 has a plurality of identical second trapping zones 18 distributed around the first trapping area 15 so that each second microdrop trapped by one of the second trapping zones 18 is in contact with the first microdrop 20.
• the second trapping areas 18 may be evenly distributed around the first trapping area 15 as illustrated. But it can be otherwise.
• the capillary trap 12 has a plurality of first trapping zones 15 arranged so that the first Microgoutts trapped by the latter are each in contact with at least one other first microdrop and a plurality of second trapping zone 18.
• the capillary trap 12 has two first contiguous trapping zones 15 and two second trapping zones, one contiguous to each first trapping area.
• the capillary trap 12 may have a plurality of second trapping areas 18a and 18b of different shapes.
• the second trapping zones may be intended to receive second microdrops 25a and 25b different.
• the capillary trap has a second trapping area 18a forming a single cavity with the first trapping area 15 and a second trapping area 18b disjoint from the first trapping area 15.
• the second trapping areas 18a and 18b exert different trapping forces on the same microdrop.
• the second trapping area 18a is intended to trap a second microdrop 25a of greater diameter than that of the second microdrop 25b trapped by the second trapping area 18b.
• the invention is not limited to the shape examples of the capillary trap 12 described above.
• the capillary trap 12 can take different forms, in particular depending on the desired application. Figures 12 to 45 illustrate possible forms.
• the first trapping area 15 may be polygonal in shape, including square, and the second trapping area 18 of triangular shape and contiguous to one side of the square.
• the second trapping area 18 is contiguous at an angle of the square forming the first trapping area 15.
• the second trapping zone 18 is of rectangular shape, in particular square.
• the second trapping area 18 can widen towards the first trapping area 15 and have opposite walls 27 and 28 presenting a curved profile in a plan view, the second trapping area 18 being tilting towards its end.
• the capillary trap 12 may have a heart shape, the lobes of the heart forming two first trapping zones 15 and the tip of the heart forming the second trapping zone 18.
• the first trapping area 15 may be pentagonal in shape with the second trapping area 18 extending from a corner of the pentagon.
• the capillary trap 12 may be hexagonal shaped, with the second trapping area 18 extending from one side of the hexagon.
• the first trapping area 15 may be square in shape, as shown in Fig. 19, or circular in shape, as shown in Fig. 20, and the second trapping area 18 may be circular in shape.
• the second trapping area 18 is triangular in shape but connected to the trapping first 15 by one of its corners, as shown in Figure 21.
• the second trapping area 18 thus widens outwardly.
• the second trapping area 18 is of polygonal shape, in particular hexagonal, as illustrated in FIG. 22.
• the capillary trap 12 may comprise two second trapping zones 18 contiguous to the first trapping area 15 at opposite angles thereof, as illustrated in FIG. 33.
• the capillary trap 12 may have a first oval-shaped trapping zone 15 and two second triangular trapping zones 18 contiguous to the first trapping zone and extending facing one of the trapping zones. other side of the latter from the long sides of the oval.
• the second trapping zones 18 are of the same shape as that of FIG. 34.
• the capillary trap 12 may have two second trapping zones 18 that are not equidistributed around the first trapping area 15 but forming between them a separation angle ⁇ .
• the first trapping area 15 may be of rectangular shape and the second square trapping area 18 contiguous to the first trapping area 15 by a small side of the rectangle. Depending on the size of the first microdroplets 20, the first trapping area 15 may then trap a single first microdrop 20, as shown in FIG. 25, or a plurality of first microdrops 20, as shown in FIG.
• the second trapping area 18 may be contiguous to the first trapping area 15 by one of the long sides of the rectangle.
• the first trapping area 15 may be oval shaped and the second trapping area 18 attached thereto from its long side as shown in Fig. 28 or its short side as shown in Fig. 29.
• the capillary trap may comprise a plurality of second trapping zones 18, at least two of which are different, in particular by their sizes, as illustrated in FIG.
• the capillary trap 12 has a form of gourd squash whose base forms the first trapping zone 15 and the head forms the second trapping zone 18.
• the capillary trap 12 is of triangular shape, the part near the wider base forming the first trapping zone 15 and the tip forming the second trapping zone 18.
• the first trapping zone 15 is square in shape and the capillary trap 12 has two second trapping zones 18 of the same square shape each contiguous with one of their corner at one corner of the first zone. trapping 15.
• the capillary trap 12 has three second trapping zones 18, the first and the second trapping zones 15 and 18 possibly having all the forms described above.
• the capillary trap 12 has a plurality of second trapping zones that differ in their shapes.
• one of the second trapping areas 18a is of triangular shape and the other of the second trapping areas 18b is of rectangular shape, the rectangle being long enough to form a plurality of second trapping areas and trapping a second microdrop chain 25, as shown in FIG. 42, where the second trapping zones may have the shapes described above.
• first trapping area 15 and the second trapping area 18 may be of constant height throughout their width.
• the first trapping zone 15 has, in cross-section, an edge inclined toward the bottom of the cavity.
• the second trapping zone 18 has a wall inclined towards the first trapping area. This makes it possible in particular to keep the second microdrop 25 in contact with the first microdrop 20.
• the first and second trapping zones have at least one inclined wall.
• the capillary trap 12 is formed at least partially by a cavity of the upper wall 7 of the microfluidic system.
• the capillary trap 12 is formed at least partially by a cavity of one of the side walls of the microfluidic system.
• the capillary trap is formed both by a cavity of the lower wall 8 and of the upper wall 7.
• the capillary trap is anisotropic and comprises a plurality of trapping zones having the same trapping force, so that the capillary trap traps a plurality of identical microdrops in its different zones.
• the capillary trap 12 may be of substantially triangular shape and trap, depending on the size of the microdroplets, 3 or 4 identical microdrops.
• the capillary trap 12 has a star shape with five branches and can trap 5 or 6 identical microdrops.
• the channel 9 for circulating the microdroplets may comprise a plurality of capillary traps 12.
• the channel 9 may comprise a two-dimensional trapping chamber 30 in which the capillary traps 12 are distributed spatially in two table or matrix spatial directions, as illustrated in FIG. 49.
• the capillary traps 12 are arranged at equal distances from each other in the form of a plurality of rows, but it may be otherwise. They can be arranged according to any scheme, periodic or not.
• the number of capillary traps 12 in the trapping chamber 30 can range from one per chamber to several thousand per cm 2 .
• the distance p defined between the centers of gravity of the capillary traps 12 is preferably greater than or equal to the size of the larger microdroplets intended to be trapped, in particular greater than or equal to the apparent diameter, seen from above, of a first drop. confined in the channel between the walls 7 and 8 outside the capillary traps, for example between 20 ⁇ and 1 cm.
• the number of capillary traps may be greater than or equal to 200 capillary traps per cm 2 , more preferably 2000 capillary traps per cm 2 . So we can realize the controlled combination of microdroplets in hundreds or even tens of thousands of traps in parallel in the same trapping chamber 30.
• Capillary traps 12 may be as previously described.
• the capillary traps 12 can all be identical.
• At least two capillary traps 12 may be different, in particular by their shapes, sizes, heights or orientations, or by the number, shape, height or orientation of the first (s) and second (s) zones of entrapment 15 and 18. This makes it possible to have different conditions depending on the capillary trap 12.
• the channel 9 can be one-dimensional and comprise a row of capillary traps 12 distributed along its length.
• the invention is not limited to the microfluidic system forms described above.
• the microfluidic system can take different forms, in particular depending on the desired application.
• the channel 9 is filled with a fluid in which the microdroplets are immiscible.
• This fluid can be stationary or in motion. When the latter is in motion, the fluid flow is preferably oriented along fluid circulation lines (not shown) and flows from a fluid inlet 31 to a fluid outlet 32.
• Microdroplets are, for example, aqueous microdroplets in an oily liquid or microdroplets of oil in an aqueous liquid.
• the first and second microdrops 20 and 25 have diameters Di and Z1 ⁇ 2 of the order of a micrometer, especially between 20 and 5000 ⁇ .
• the first microdrops 20 are preferably different from the second microdrops 25, in particular by their sizes and / or their compositions.
• the first microdrops 20, respectively the second microdrops 25, can form a panel of microdrops of which at least a certain number are different.
• the first and / or second microdrops 20 and 25 may comprise an identification compound allowing their identification before, during and / or after the coalescence of the first (s) and second (s) microdroplets 20 and 25 in contact.
• This or these identification compounds may be for example beads or particles in a number, compounds of various colors or shapes or compounds emitting a colorimetric or fluorescence signal proportional to their concentration in the microdrop.
• a panel of first microdrops and / or a panel of second microdrops comprising a compound of interest in different concentrations and / or different compounds of interest, it is thus possible to associate the position of a first and / or second microdrop in the trapping chamber with its composition in order to establish a map of the microdrop trapped in the trapping chamber.
• the identification compound (s) of the first microdroplets interact with one or more identification compounds of the second microdroplets so as to allow identification of the microdrop obtained after melting.
• melting microdrops can result in a chemical reaction of which at least one of the products can be identified.
• the channel 9 is filled with a fluid containing a surfactant.
• a surfactant allows the stabilization of microdrops and the reproducibility of their formation.
• the surfactants also make it possible to prevent spontaneous coalescence of the microdroplets in case of contact during their transport from the production device to the capillary traps or in the capillary traps.
• the surfactant is, for example, aqueous microdroplets one compound selected from the PEG-di-Krytox in a fluorinated oil or SPAN ® 80 in mineral oil.
• the surfactant is, for example, for microdroplets of sodium dodecyl sulfate oil.
• the microdroplets are stabilized by other means, in particular the microdrops can be gelled, or stabilized by adsorption of amphiphilic nanoparticles as described in the article by Pan, M., Rosenfeld, L., Kim, M., Xu, M., Lin, E., Derda, R., & Tang, SKY (2014). Fluorinated Pickering Emulsions Impede Interfacial Transport and Rigid Interface for the Growth of Anchorage-Dependent Cells. Applied Materials & Interfaces, 6, 21446-21453, incorporated herein by reference.
• FIG. 1 An example of a method of handling the first and second microdroplets is illustrated in FIG.
• step 40 the first microdrops 20 are produced.
• Numerous methods have already been proposed for forming such first microdrops in a mobile phase. Examples of the process can be cited: a) process known as "flow-focusing” described for example in SX. Anna, N. Bontoux and HA Stone, "Training of dispersions using 'Flow-Focusing' in microchannels," Appl. Phys. Lett. 82, 364 (2003), the content of which is incorporated herein by reference,
• step emulsification process described for example by R.
• microdroplets of substantially equal size.
• the dimensions of the microdroplets obtained can be controlled by modifying the formation parameters of the microdroplets, in particular the speed of circulation of the fluids in the device and / or the shape of the device.
• the production of the first microdrops can be made on the same microfluidic system as the process or on a different device.
• the first microdrops 20 can be stored in one or more external containers before being injected into the microfluidic system.
• These first microdrops 20 may all be identical or some of them may be of different compositions, concentrations and / or sizes.
• the latter can be conveyed to the capillary trap 12 by entrainment by a flow of a fluid and / or by slopes or reliefs in the form of rails of the channel 9.
• the addition of rails can make it possible to optimize the filling of the capillary traps 12, selectively, for example in combination with the use of an infra-red laser, as described by E. Fradet, C. McDougall, P. Abbyad, R. Dangla, D. McGloin, and CN Baroud, in "Combining rails and anchors with laser forcing for selective manipulation within 2D droplet arrays.” Lab Chip, vol. 11, no. 24, pp. 4228-34, Dec. 2011.
• transport of the storage microfluidic system can be done directly via a tube connecting for example the production system and the trapping system or by suction and injection with a syringe.
• the first microdroplets 20 are entrained in the microfluidic system so that the driving force that they undergo is less than the trapping force of the first trapping zones 15 on the first microdroplets 20.
• the first microdrops 20 are then trapped in step 42, in the capillary traps 12, in particular in the first trapping areas 15. If the entrainment flow exerts on the first microdroplets 20 a driving force on the first microdrops 20 greater than the trapping force of the traps. second zones of trapping 18, the latter are not trapped in the second trapping areas 18 which remain free.
• the first microdroplets 20 may be trapped in the second trapping zones 18, in particular if the sizes of the traps and drops are adapted. It is then possible to drive them out of the latter by increasing the driving force applied to all the first microdrops 20, for example by increasing the speed of the fluid flow or, when there is none, by adding a flow of fluid in a step 44.
• the first microdrops 20 may be formed by a method called "breaking drops in capillary traps" described for example in the international application WO 2016/059302, the content of which is incorporated herein by reference. In this case, the first microdroplets 20 are directly formed in the first trapping zones 15.
• step 46 the second microdrops 25 are produced. This step is shown after step 44 but could take place before.
• the second microdrops 25 may be produced and introduced into the microfluidic system as described above in relation to the first microdrops 20.
• the second microdroplets 25 are entrained in the microfluidic system so that the driving force they undergo is less than the trapping force of the second trapping zones 18 on the second microdroplets 25.
• the second microdrops 25 are then trapped in step 48, in the second trapping zones 18.
• the latter exerts on the first microdrop trapped in the first trapping zones 15 a driving force, preferably less than or equal to the trapping force of the first trapping areas 15 on the first microdrops 20 so that the latter remain trapped.
• the method may comprise a step of first measurement of the state of the system.
• This measurement can be a simple image, or for example a colorimetric, fluorescence, spectroscopic (UV, Raman) or temperature measurement.
• This measurement may be particularly useful in the context of the use of different first and / or second microdrop panels comprising an identification compound as described above. When several microdrops are in contact in the same trap, it is possible to merge them in a controlled manner to mix their contents in step 50. This coalescence can be selective or not.
• the microfluidic system In particular in the trapping chamber 30, the latter is perfused with a fluid free of surfactant.
• concentration of surfactant in the fluid of the microfluidic system decreases, which makes it possible to shift the surfactant adsorption balance at the interface towards the desorption.
• Microdroplets lose their stabilizing effect and fuse spontaneously with the microdroplets with which they are in contact.
• the microfluidic system is perfused with a fluid containing a destabilizing agent.
• the destabilizing agent is, for example, 1H, 1H, 2H, 2H-perfluorooctan-1-ol in a fluorinated oil in the case of aqueous microdots.
• all the microdroplets in contact in the microfluidic system, in particular in the trapping chamber 30, are fused together by bringing an external physical stimulus, such as mechanical waves, pressure waves, a temperature change or a field. electric.
• electrodes 35 may be placed on either side of the trapping chamber so as to fuse all the microdroplets in contact therebetween.
• an infra-red laser can be used, as described by E. Fradet, P. Abbyad, M. H. Vos, and C. N. Baroud, in Parallel measurements of reaction kinetics using ultralow-voizis. Lab Chip, Vol 13, No. 22, pp. 4326-30, Oct. 2013, where localized electrodes 37 at the microdropper interfaces between trapping zones can be activated, as shown in Fig. 51. , or mechanical waves can be focused at one or more points.
• the invention is not limited to the coalescence examples described above. Any method for destabilizing the interface between two microdrops in contact can be used to fuse the microdrops.
• the capillary trap (s) 12 have a plurality of second trapping zones 18, the position of the second trapping zone (s) 18 by With respect to the direction and direction of the driving force exerted on the second microdrops 25 may allow selective trapping.
• second microdroplets 25m are supplied with a driving force oriented along the direction Fi according to a step 48a. If the trapping forces in the second trapping areas 18m and 18v are sufficiently low, a second microdrop 25m is trapped only in the second trapping area 18m upstream of the first trapped microdriple relative to the direction Fi.
• the trapping forces in the second trapping zones 18m and 18v are sufficiently strong to each trap a second microdrop 25m regardless of the direction of the driving force exerted on the second microdrop 25m in step 48a, the fact that the second microdrop 25m upstream with respect to a drive force oriented in the direction F 3 is retained better than that downstream when such driving force is applied can be used to release s tively the second microdroplet 25m trapped downstream in a step 52.
• the first microdroplet 25m can deform and press the first microdroplet 20 trapped in the first trapping region 15, so it takes a larger force strong to release it only to release the second microdrop 25m downstream. It is then possible to trap in the second trapped 18v trapping zone a second microgout 25v different from the second microdrop 25m already trapped in step 48b.
• the method comprises the following steps, for a capillary trap presenting two zones. trapping:
• the trapping force F1 exerted by the first zone on the first microdrop being greater than the force Ft1 of hydrodynamic drag exerted by the flow on the first microdrop, such that so that the latter remains trapped in the first zone, the drag force Ft1 being between Fl and F2, F2 designating the trapping force exerted on the first microdrop by the second capillary trap area,
• F3 with F3 preferably lower than F1, F3 being the trapping force exerted by the second trap area on the second microdrop.
• the drag force Ft2 is less than Fl.
• the second trapping zones 18 of the different capillary traps 12 in the same microfluidic system may have different properties, especially different sizes. This makes it possible, for example, to trap second different microdroplets in different capillary traps 12 in order to obtain different microdroplets. For example, for a given concentration of an element included in the second microdroplets, the quantity of its elements contained in a second microdrop depends on the size of said second microdrop. Thus, by producing second trapping zones 18, for example, of different sizes in the same chamber 30, it is possible to selectively trap second microdrops 25 of different sizes, at each size of second trapping area 18 corresponding to a second microdrop size 25.
• Figure 55 illustrates three capillary traps 12a, 12b and 12c having second trapping areas 18a, 18 and 18c of different sizes.
• the first trapping zones 15 are filled with first microdroplets 20.
• Second microdrops 25a, 25b and 25c, all of the same concentration but of different sizes are made.
• the second trapping areas 18a, 18b and 18c each correspond to a second microdrop size, the second microdroplets 25a, 25b and 25c are then trapped in the second trapping area 18a, 18b or 18c which corresponds best to them.
• the capillary traps 12a, 12b and 12c by trapping forces of the second trapping areas 18a, 18b and 18c with respect to the larger of the second growing microdroplets 18a in the direction of the driving force of the second microdroplets. , especially in the direction of fluid flow, in the microfluidic system.
• the second microdroplets 25a, 25b and 25c first meet the second trapping areas 18c in which only the second smallest microdroplets 25c are trapped, then the second trapping areas 18b in which only the second microdroplets 25b are trapped and finally the second trapping areas 18a in which the second largest microdroplets 25a are trapped.
• microdroplets 25a, 25b and 25c are different by another of their parameters, in particular they comprise different elements.
• Steps 46, 48 and 50 may be repeated as shown in Fig. 56 to effect sequential coalescence.
• the microdrop obtained after step 50 becomes the new first microdrop 80 having the volume of the sum of the volumes of the first microdrop or microdroplets 20 and the second or microdroplets 25 fused into the capillary trap 12.
• the second trapping zone or traps 18 are again free, it is possible to bring one or more third microdroplets 58 identical or different to the initial microdrop or second microdroplets to achieve a new coalescence and obtain a new microdrop 90 becoming itself the new first microdrop and so on.
• the microdrop will grow due to the addition of the different volumes of coalesced microdroplets to be so large that it prevents the trapping of a new microdrop in the second trapping area 18.
• the maximum number of coalescence achievable in the same capillary trap 12 depends on the volume of successive coalesced microdroplets.
• coalescing step consists in removing the surfactant from the external phase or destabilizing it chemically
• a stabilization step may be necessary between the different coalescences.
• first microdrop trapped in a first trapping area of a capillary trap 12 contains cells (for example, bacteria, yeasts or mammalian cells)
• sequential coalescence may allow the culture medium to be repeated several times by sequentially fusing.
• second microdrops 25 containing a culture medium at predetermined times.
• a first microdrop 20 containing a spheroid of mammalian cells and trapped in a capillary trap 12 is fused every 6 hours with a second drug microdroplet.
• the capillary traps 12 in the trapping chamber 30 may be different and their locations may be controlled. It is possible, for example, to have capillary traps 12 having a different number of second trapping zones.
• the trapping chamber 30 may comprise capillary traps 12a, 12b, 12c and 12d having respectively one, two, three and four identical second trapping zones 18 distributed around a first trapping area 15 If the first microdrops 20 are identical and comprise a compound of interest, it is possible to obtain in the microfluidic chamber microdroplets 100, 105, 110 and 115 forming a spatial gradient of four concentrations of compound of interest, represented here by different levels of gray, after coalescence with the second trapped microdroplets which may for example contain a diluent. Panels of microdroplets
• microdrop panels with different compounds and / or different concentrations in a chamber containing capillary traps 12 as described above allows many applications.
• the first microdrops 20 form a panel of microdrops containing a first compound in 20 different concentrations.
• the second microdrops 25 contain a second compound in 10 different concentrations.
• microdrops are static also facilitates the obtaining of kinetic data.
• economy in reagents is also obtained by using very small volumes in microdroplets.
• the trapping chamber 30 may have an area greater than 2 cm 2 which further greatly increases the number of different reactions that can be carried out in parallel in a microfluidic system.
• the microdrops may then comprise one or more identification means. This allows for example to be able to measure the concentration of the final product, for example by fluorescence or spectroscopy.
• first and / or second microdrops 20 and / or 25 may contain proteins, enzymes, cells at various concentrations.
• microfluidic system and the method described above can be used to study the crystallization of proteins. Indeed, obtaining a crystal from a purified solution of protein is an essential step for determining its three-dimensional structure since this makes it possible to obtain an X-ray diffraction pattern.
• a trapping chamber 30 with several capillary traps 12 each making it possible to trap a first and a second microdrop 20 and 25 makes it possible, for example, to have a first microdrop panel comprising a saline solution in different concentrations and a second microdrop panel comprising a protein of interest in different concentrations.
• a first microdrop panel comprising a saline solution in different concentrations
• a second microdrop panel comprising a protein of interest in different concentrations.
• first microdrops 20 having an element of interest in identical concentration throughout the trapping chamber and merging the latter with second microdrops 25 from a panel of microdrops comprising a titrant species in different concentrations it is possible to perform a titration of the species of interest contained in the first microdrops 20.
• This application can be particularly interesting in the case of expensive reagents or available in small quantities.
• cancer cells can be cultured in individualized form or as spheroids in first microguides trapped in each of the first trapping area of the capillary traps 12, and after a few days of culture, it is possible to have them coalesced in each trap with a second microdrop containing a drug to be screened, the second microdrops being from a panel of microdrops containing different drugs.
• a panel of second microdrops containing one or more antibiotics in different concentrations can be created and fused into the trapping chamber 30 with first microdrops containing bacteria.
• the first microdrops 20 may have a bacterium in different concentrations. This allows to explore a space with 3 parameters.
• micro fluidics can be very advantageous in the context of rare samples such as biopsies.
• the microfluidic system can for example be used in the context of personalized medicine and cancer treatment.
• tumor cells of a biopsied patient can be cultured, for example as spheroids in first trapped microdroplets, and subjected to different drugs at multiple concentrations via delivery of the drugs.
• the most effective drug and its concentration for a particular patient can be determined using only one entrapment chamber 30 and a second one. minimal number of cells from the biopsy.
• the method as described above can be used to fuse microdrops containing cells of different or different cell types to accurately form microtissues.
• the capillary trap may be as illustrated in Figures 5A and 5B.
• the first trapping area 15 has a height such that its volume is greater than that of the first microdrop so that the first microdrop trapped in a non-flat bottom, especially convex, as shown in Figure 5A.
• the cells contained in said first microdrop 20 slide along the interface of the latter during their sedimentation to aggregate at the bottom of the first trapped microgout and form a spheroid, as described in the international application WO 2016/059302, incorporated herein by reference.
• a first microdrop 20 may contain cells of a first cell type in a liquid medium and be trapped in the first trapping zone 15 of said capillary trap 12, after one day of immobilization, spontaneously forming a first spheroid by sedimentation of the cells .
• the microfluidic system is free of fluid flow near the capillary trap during the formation of the first spheroid so that the liquid of the first microdrop 20 is not driven in motion.
• a second microdrop 25 containing cells of a second cell type in a liquid medium can be trapped in the second trapping area 18. After coalescence of the first and second microgoutts, a culture of cells of two different types is obtained, the architecture of which depends in particular on the experimental conditions.
• the cells of the second microdrop 25 mix, after coalescence, with the contents of the first microdrop 20 and then sediment to directly reach the spheroid of cells of the first cell type.
• the coalescence of the two microdrops 20 and 25 results in the fusion of the first and second spheroids.
• the two cell populations are compartmentalized.
• the cells of the second cell type will no longer be able, after coalescence of microdroplets 20 and 25, to come into direct contact with the first spheroid because of the presence of the gel.
• mammalian cells can not pass through an agarose matrix at 0.9% by weight. The two groups of cells can then communicate together only paracrine.
• capillary trap 12 trapping a first and a microdrop 20 and 25 but it is possible to obtain more complex microtissus architectures with a capillary trap 12 for trapping more than two microdrops and / or processes as previously described consisting of sequentially coalescing several microdroplets by varying or not the orientation of the fluid flow. It is also possible to use a plurality of capillary traps as described above to form a plurality of microtissues in parallel.
• microtissus formation can make it possible to create in vitro microtissus with a controlled architecture to mimic very faithfully the conditions encountered in vivo.
• the different cell types are often arranged in tissue according to a specific architecture that is important to recreate a function at an organ.
• the latter can also be used for transplantation on a patient.
• insulin-producing beta cells, glucagon producers, and beta cells may be combined to create Langerhans islets for transplantation into a patient's pancreas to cure diabetes.
• hepatocytes and stellate cells could be associated with liver transplantation.
• the method as described above can also be used to create multilayer gel microdroplets.
• the capillary trap 12 may be as described above and comprise a first trapping zone 15 and a second trapping zone 18.
• a first microdrop 20 containing a first gelling medium can be trapped in the first entrapment zone 15, and then the first gelling medium can be gelled to form, as illustrated in step 54, a microdroplet 200 of a first gel.
• the first gelled microgout 200 can then be fused, according to step 58, with a second microdrop 25 containing a second gellable medium and trapped in step 56 in the second trapping zone 18.
• the second gellable medium can be gelled to form an outer layer 202 of a second gel on the first gel. This operation can be repeated several times sequentially to form a microdrop with a core of the first gel and a plurality of successive outer layers of the other gels.
• the second gelling medium contained in the second microdrop 25 is gelled to form a microdrop 204 of a second gel before fusing with the first microdrop.
• the two microdrops When the two microdrops are fused, they retain their shape and position before melting and form a gelled microgout 210 of shape dependent on the shape, arrangement and number of trapping areas.
• the method can also make it possible to obtain spheroids encapsulated in biological hydrogels.
• the capillary trap may be as illustrated in Figures 5A and 5B.
• the first trapping area 15 has a height such that the first microdrop trapped at a non-flat bottom, in particular a convex bottom, as illustrated in FIG. 5A so that the cells contained in said first microdrop 20 slide along the interface of the latter during their sedimentation to aggregate at the bottom of the first trapped microgout and form a spheroid, as described in the international application WO 2016/059302, incorporated herein by reference.
• a first microdrop 20 can contain cells of a first cell type in a liquid medium and be trapped in the first trapping zone 15 of said capillary trap 12, after one day of immobilization spontaneously forming a spheroid by sedimentation of the cells.
• the microfluidic system is free of fluid flow in the vicinity of the capillary trap during spheroid formation so that the liquid of the first microdrop 20 is not driven in motion.
• a second microdrop containing one of the biological hydrogels mentioned above in high concentration can be trapped in the second trapping zone 18. Once this second microgout is trapped, the two microdroplets are fused immediately so that the biological hydrogel, still liquid, mixes with the first microdrop that contains the spheroid.
• the gelation then takes place and the spheroid is encapsulated in an extracellular matrix representative of the biological conditions encountered in vivo.
• This spheroid encapsulation technique can be combined with the microtissue formation technique previously described to provide more complex microtissue architectures.
• the trapping chamber 30 used is 2 cm 2 and contains 393 identical capillary traps similar to that of FIGS. 1A, 1B and 2 and having the following dimensions:
• the capillary traps 12 are distributed according to a matrix as illustrated in FIG. 49.
• Microdots have food coloring. Drops of 1 ⁇ , with five different colors ranging from blue to yellow through green were formed by the technique of "micro-segmented flows". These drops of 1 ⁇ were fractionated into many first monodisperse nanodisperse microdroplets using a slope (method described in point f) above). These first microdrops 20 of different colors were then mixed to form a first panel of microdrops having different colors and then injected into the trapping chamber 30 containing the capillary traps 12. The size of the first microdrops 20 was adjusted to occupy them fully. the first trapping areas 15. The second trapping areas 18 remain empty.
• a matrix of pairs of first and second microdroplets 20 and 25 as shown in FIG. 60a) is then obtained.
• the first and second microdroplets 20 and 25 in contact are fused by infusing the entrapment chamber 30 with HFE-7500 containing 1H, 1H, 2H, 2H-perfluorooctyl-1-ol at a concentration of 20% by volume.
• the colors of the two microdrops in contact in each of the capillary traps mix to take one of the possible colors according to the color of the initial microdrops, as can be seen in Figure 60b).
• a matrix of microdroplets of 25 different colors is then obtained.
• the microfluidic system comprises a trapping chamber 30 having a matrix of capillary traps as illustrated in FIGS. 5A and 5B having the following dimensions:
• Rat liver cells (H4IIEC3) were first encapsulated in first microdrops 20.
• the first microdroplets were trapped in the first trapping areas and, after sedimentation, the cells pooled at the bottom of each drop to form a first spheroid 130.
• second microdrops 25 were trapped in the second trapping zones 18, as can be seen in FIG. 61 A.
• These also contain H4IIEC3 cells but, unlike the former, they were stained red with a fluorescent marker (CellTracker Red®).
• first and second microdroplets 20 and 25 were kept in the state for one day so that the red cells of the second microdroplets 25 gather and form a second spheroid 135 at the bottom of each second microdrop 25.
• the first and second microdroplets in contact were then fused by infusing the chamber with HFE-7500 (fluorinated oil) containing 1H, 1H, 2H, 2H-perfluorooctan-1-ol at a concentration of 20% by volume.
• HFE-7500 fluorinated oil
• the second spheroid 135 sediments and contacts the bottom of the first microdrop with the first spheroid 130 in a new first microdrop 140, as can be seen in FIG. 61B.
• These two spheroids in contact will adhere to each other and merge to form a single new spheroid two parts 130 and 135 clearly identifiable by fluorescence imaging (cells from the second spheroid will continue to appear red in the fused spheroid).
• the chamber is then infused with oil containing surfactant to restore the stability of the new first microdrops for further experimentation.
• the first microdroplets were then kept one day in culture to allow the cells to adhere to each other to form a spheroid by first microdrops 20.
• the first microdrops 20 are then gelled by applying a temperature of 4 ° C. 30 min.
• acetaminophen has been solubilized at high concentration in culture medium. Fluorescein at high concentration was added to this solution. The solution obtained was diluted in different concentrations with pure culture medium to form drops of ⁇ ⁇ at different concentrations. These drops were then fractionated into second microdrops using a slope and then mixed before being injected into the trapping chamber 30 which contains the first microdroplets 20. These second microdrops 25 were smaller than the first microdrops 20 and were trapped in the second trapping areas 18.
• the chamber was perfused with HFE-7500 (fluorinated oil) containing 1H, 1H, 2H, 2H-perfluorooctan-1-ol at a concentration of 20% by volume to fuse the microdrops in contact.
• HFE-7500 fluorinated oil
• Acetaminophen then diffused through the gelled agarose to act on the cells.
• the oil that separates the microdroplets from each other is replaced by an aqueous phase as described in the international application WO 2016/059302 for coloring spheroids with fluorescent viability markers present in the aqueous phase.

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