Patent Grant
11179722
The present invention relates to a microfluidic method for handling several microdrops in at least one capillary trap of a microfluidic system. The invention also relates to a microfluidic device for carrying out said method.
Trapping of microdrops circulating in one or more microchannels in traps of approximately circular or oval shape, each trapping zone being dimensioned for trapping a predefined number of microdrops, is known from patent application FR 2 950 544.
Trapping and fusing of microdrops of roughly identical or of different sizes in a shallow trap of approximately circular shape of a microfluidic system, the latter being two-dimensional and having a plurality of traps, is also known from E. Fradet, C. McDougal, P. Abbyad, R. Dangla, D. McGloin, and C. N. Baroud, “Combining rails and anchors with laser forcing for selective manipulation within 2D droplet arrays.” Lab Chip, Vol. 11, No. 24, pp. 4228-34, December 2011, and J. Tullis, C. L. Park, and P. Abbyad, “Selective Fusion of Anchored Droplets via Changes in Surfactant Concentration.” Lab Chip, 2014. The microdrops trapped in one and the same trap are different.
Traps of this kind do not allow precise manipulation and/or control of the trapped microdrops, notably to adapt the traps to microdrops of different sizes, nor trapping of the microdrops in a spatially predefined manner.
E. Fradet, P. Abbyad, M. H. Vos, and C. N. Baroud, “Parallel measurements of reaction kinetics using ultralow volumes.” Lab Chip, Vol. 13, No. 22, pp. 4326-30, October 2013, describes a shallow trap having two identical zones of approximately circular shape that overlap partially to make the trap goggle-shaped. Each of the two zones makes it possible to trap one microdrop. The shape of the trap makes it possible to keep the two trapped microdrops in contact with one another so as to fuse them into a single microdrop. A trap of this kind is limited to handling two microdrops of roughly identical sizes and is not suitable for treating a large number of microdrops, which reduces the possible applications.
Moreover, a C-shaped trap for immobilizing and fusing two microdrops is known from A. M. Huebner, C. Abell, W. T. S. Huck, C. N. Baroud, and F. Hollfelder, “Monitoring a Reaction at Submillisecond Resolution in Picoliter Volumes.”, Anal Chem. 2011 Feb. 15; 83(4): 1462-8. The trap is formed by projecting reliefs that block the microdrops in their flow. However, manipulation of the microdrops is limited notably owing to the shape of the traps, and retaining the microdrops in the traps requires the presence of an oriented stream of fluid in a precise direction.
Application WO 2016/059302 describes a method for handling microdrops in a microfluidic system comprising the step consisting of trapping the microdrops in a capillary trap and at least partially gelling the microdrops or their environment. The capillary trap is able to receive several microdrops in its depth, which is greater than the diameter of the trapped microdrops. However, depth-wise manipulation of the microdrops, notably of those that are trapped, is limited.
Application US 2015/0258543 proposes a method that allows different fluids to be brought into contact to obtain a reaction between them and allow analysis of the kinetics of this reaction. A microfluidic circuit is disclosed there, which is able to bring the microdrops, one at a time, into cavities serving as capillary traps. Two microdrops of different volumes can be brought into contact and received in an 8-shaped cavity. The dimensions of the two trapping zones, each trapping zone corresponding to a loop of the 8, correspond to the dimensions of the microdrops that are to be housed there. There is an energy barrier between these different trapping zones, associated with the shape of the cavity. For example, a microdrop supplied from the right will remain in the trapping zone on the right, on account of the 8-shape of the cavity.
Application US 2010/0190263 describes an actuator of droplets having hollowed regions separating two substrates. These substrates include electrodes for transporting the droplets to the hollowed regions. The purpose of the actuator is to form and retain a gas bubble in these hollowed regions.
The articles Dangla, R., Lee, S. & Baroud, C. N. Trapping microfluidic drops in wells of surface energy. Phys. Rev. Lett. 107, 124501 (1-4) (2011) and Yamada, A., Lee, S., Bassereau, P. & Baroud, C. N. Trapping and release of giant unilamellar vesicles in microfluidic wells. Soft Matter 10, 26-28 (2014) describe the physics associated with the trapping of microdrops, subjected to fluid flow, in a microchannel. The trapping force may result from the shape and size of the trap, the depth of the microchannel, the size of the microdrops, and the physical and physicochemical properties of the fluids present, such as viscosity, surface tension, etc.
There is therefore a need for a method for handling microdrops allowing the trapped microdrops to be controlled easily and to be trapped in a spatially predefined manner. There is also a need for a method allowing sequential manipulation of the microdrops.
I. First Aspect—Method of Manipulation
For this purpose, according to a first of its aspects, the invention proposes a method for manipulating at least one first microdrop and at least one second microdrop in a microfluidic system comprising a capillary trap having a first trapping zone and a second trapping zone, said method comprising the steps consisting of:
trapping the first microdrop in the first trapping zone, and
(ii) trapping the second microdrop in the second trapping zone,
the first and the second trapping zone being arranged in such a way that the first microdrop and the second microdrop are in contact with one another,
the first and the second trapping zone being configured in such a way that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
“Microfluidic system” denotes a system involving the transporting of at least one product, and which comprises, on at least one of its portions, a section, at least one dimension of which, measured in a straight line from one edge to an opposite edge, is less than a millimeter.
“Microdrop” means a drop having a volume less than or equal to 1 μl, better still less than or equal to 10 nl. The microdrop may be liquid, gaseous or solid.
“Capillary trap” means a spatial zone of the microfluidic system allowing 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, notably hollowed reliefs, and/or by one or more local modifications of the surface in contact with the microdrops, notably one or more local modifications of the affinity of the surface with at least part of the contents of the microdrop.
“The trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different” means that if the first microdrop is trapped in the first trapping zone only, it will be retained in the latter by capillarity with a trapping force that is different from the trapping force that the second trap zone would exert on this same first microdrop on its own. Thus, it is easier to release the first microdrop from the trapping zone that exerts the smallest trapping force. The same reasoning may be applied to the second microdrop. The trapping force of a trapping zone depends notably on its shape, its surfaces in contact with the microdrop and/or the properties, notably the dimensions, of the microdrops to be trapped.
The fact that the capillary trap has two zones having different trapping forces exerted on one of the microdrops makes it possible to have both selectivity of the trapped microdrops and spatial selectivity, and notably to avoid the first microdrop occupying the second trapping zone, thus preventing the second microdrop from being trapped in this second trapping zone. This is particularly advantageous when a plurality of first and second microdrops is introduced into the microfluidic system.
The fact that the first and second microdrops are in contact allows them either to interact, or to coalesce.
Preferably, the first microdrop is trapped in the first trapping zone with a trapping force that is greater than what would be exerted by the second trapping zone on the first microdrop. Thus, the first microdrop is preferably trapped by the first trapping zone.
As a variant, the second microdrop is trapped in the second trapping zone with a trapping force that is smaller than what would be exerted by the first trapping zone on the second microdrop. In this case, the first and second microdrops are preferably introduced and trapped in the microfluidic system sequentially.
Preferably, the first microdrop is trapped by the first trapping zone in the microfluidic system before the second microdrop is trapped by the second trapping zone in the microfluidic system. Thus, when the second microdrop is introduced, it cannot occupy the first trapping zone as the latter is already occupied by the first microdrop.
An entraining force greater than the trapping force of the second trapping zone and less than or equal to the trapping force of the first trapping zone may be exerted in step (i) on the first microdrop. For example, the shape of the second trapping zone is selected in such a way that the trapping force of the second zone is less than the entraining force. In other words the microdrop is subjected to hydrodynamic forces owing to its entrainment that oppose its capture by the second trapping zone. The drag force exerted by the fluid conveying the microdrops 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 flow velocity. The first drop is then only trapped in the first trapping zones.
The entraining force may be exerted at least partially by
Preferably, the second microdrop is subjected to an entraining force as described in relation to the first microdrop, less than or equal to the trapping force of the second trapping zone. In the case when the entraining force is exerted on the second microdrop by an oriented stream of fluid, the force that the stream of fluid exerts on the first microdrop trapped in the first trapping zone is preferably insufficient to extract the first microdrop from the first trapping zone.
In the case when the entraining force on the second microdrop is exerted by an oriented stream of fluid, the latter may be oriented in such a way that the second microdrop can only be trapped in the second trapping zones having a particular orientation relative to the orientation of the stream, notably arranged upstream of the first trapping zone relative to the direction of the stream of fluid.
The microdrops being carried by a stream of fluid to a plurality of trapping zones, they are preferably led into the latter randomly and naturally occupy the place that is the most advantageous, from an energy standpoint, of the trapping zone. They lodge in the trapping zones by themselves. This random placement of the microdrops makes it possible to have a large number of microdrops trapped simultaneously, and increases the capacity of screening.
In embodiment examples, the method comprises the following steps:
Microdrops
Preferably, the trapping force that the first trapping zone exerts on the first microdrop is different from the trapping force that it would exert on the second microdrop and the trapping force that the second trapping zone exerts on the second microdrop is different from the trapping force that it would exert on the first microdrop. In fact, the trapping force also depends on the shape of the microdrop to be trapped in relation to the shape of the trapping zone. This facilitates sequential trapping of the first and second microdrops.
Preferably, the first trapping zone exerts a trapping force on the first microdrop that is greater than what it would exert on the second microdrop. This makes it possible to prevent the second microdrop dislodging and taking the place of the first microdrop.
The first and the second microdrop may be different, in particular of different sizes, notably the first microdrop being of larger size and/or of larger volume than the second microdrop, and/or with different contents. This guarantees that the first trapping zone exerts a trapping force on the first microdrop that is greater than what it would exert on the second microdrop; consequently, there will only be a single microdrop in the first trapping zone.
The first trapping zone may trap one or more first microdrops.
The second trapping zone may trap one or more second microdrops.
As a variant, the first and second microdrops are different in at least one of their properties, notably their viscosity and/or their interfacial tension and/or their affinity with a particular coating of at least one of the trap zones.
Preferably, the first trapping zone only traps a first microdrop and/or the second trapping zone only traps a second microdrop. In particular:
One of the first microdrop(s) or second microdrop(s) may be a microbubble of air.
Plurality of Second and/or First Trapping Zones
The capillary trap may comprise a plurality of second trapping zones, step (ii) consisting of trapping one second microdrop per second trapping zone, the first and the second trapping zones being arranged in such a way that each second microdrop is in contact with at least one of the first or of the second microdrops.
The capillary trap may comprise a plurality of first trapping zones, step (i) consisting of trapping one first microdrop per first trapping zone, the first and the second trapping zone or zones being arranged in such a way that each first microdrop is in contact with at least one of the second microdrop(s) or of the first microdrops.
Preferably, each second microdrop is joined to the or each first microdrop.
“Joined to” means that 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” means a plurality of microdrops forming a straight line or curved line in contact with one another.
The second microdrops may all be in contact with at least one first microdrop trapped in said capillary trap.
At least two second trapping zones may be configured so that the trapping forces that they would exert on one of said second liquid microdrops would be different. The second microdrops trapped by at least two second trapping zones may be different with respect to at least one of their properties, notably their largest dimension.
As a variant, all the second trapping zones of the capillary trap are identical.
Plurality of Capillary Traps
The microfluidic system may comprise a plurality of capillary traps, each comprising a first trapping zone and a second trapping zone, step (i) consisting of trapping a first microdrop in the first trapping zone of each capillary trap, step (ii) consisting of trapping a second microdrop in the second trapping zone of each capillary trap, the first and the second trapping zone of each capillary trap of the plurality of capillary traps being arranged in such a way that the first and the second microdrop trapped in said capillary trap are in contact with one another in the latter.
Each capillary trap may comprise one or more of the features described above.
All the traps of the microfluidic system may each comprise a first trapping zone and a second trapping zone arranged in such a way that the first and the second microdrop trapped in said capillary trap are in contact with one another in the latter.
As a variant, only some of the capillary traps of the microfluidic system each comprise a first trapping zone and a second trapping zone arranged in such a way that the first and the second microdrop trapped in said capillary trap are in contact with one another in the latter.
The method may comprise the step consisting of trapping a microbubble of gas, notably of air, in one of the first or second trapping zones. This makes it possible to render the trapping zone in question inactive. In fact, owing to the presence of the gas microbubble, the first or second microdrops cannot be trapped in the trapping zone in question.
Oriented Stream of Fluid
Step (ii) may comprise the substeps (ii′) consisting of trapping, under the effect of a first oriented stream of fluid, a second microdrop in one or some of the second trapping zones and (ii″) consisting of trapping, under the effect of a second oriented stream of fluid, a second microdrop in another or some others of the second trapping zones, the first and the second stream of fluid being of different orientation.
The second microdrops of steps (ii′) and (ii″) may differ notably by at least one of their properties and/or their contents. The second trapping zones of steps (ii′) and (ii″) may be identical.
Thus, it is possible, by selecting the orientation of the stream of fluid, to trap microdrops selectively in one of the two trapping zones, which allows predefined spatial positioning of the microdrops in contact with one another. It is then possible to bring a first microdrop into contact with different second microdrops in a controlled manner, notably in the context of combinatorial chemistry. It is also possible, in the context of microdrops comprising gels, to control the spatial disposition of gel microdrops in the capillary trap so as to obtain a microdrop of controlled shape and composition after fusion.
Coalescence
The method may comprise the step (iii) consisting of fusing, with the first microdrop, the or each of the second microdrops trapped in the or each of the second trapping zones. Such coalescence notably allows the contents of the two microdrops to be mixed.
Said coalescence may be selective, i.e. we may select the second microdrop or microdrops in contact with the first microdrop that we wish to fuse with the latter, notably by using an infrared laser, as is described for example in the document E. Fradet, P. Abbyad, M. H. Vos, and C. N. Baroud, “Parallel measurements of reaction kinetics using ultralow volumes.” Lab Chip, Vol. 13, No. 22, pp. 4326-30, October 2013, the contents of which are incorporated by reference, addressable electrodes arranged at the level of the capillary trap, or mechanical waves.
As a variant, the coalescence of the microdrops is nonselective, i.e. all of the second microdrops of the capillary trap fuse simultaneously with the first microdrop, notably by adding a product that promotes this coalescence to the environment of the capillary trap or application of an external physical stimulus such as mechanical waves, pressure waves, a temperature change or an electric field.
Release of the Second Microdrops
As a variant, step (iii) consists of evacuating the or at least one second microdrop trapped in the second trapping zone outside of the capillary trap. Step (iii′) may consist of applying an oriented stream of fluid, configured for exerting, on one or more second microdrops, an entraining force greater than the trapping force of the second trapping zone, the stream of fluid being configured for exerting, on the first microdrop or microdrops, an entraining force less than or equal to the trapping force of the first trapping zone, so that the first microdrop or microdrops remain trapped in the first trapping zone.
In this step, it is possible to evacuate one or more of the second microdrops from the second trapping zones, the capillary trap being configured so that, owing to its orientation, the stream of fluid exerts different forces of entrainment on the second trapping zones, the method preferably comprising step (iv) consisting of changing the orientation of the stream of fluid so as to evacuate at least one or more of the second microdrops from at least one other trapping zone. This allows selective release of the second microdrops. Thus, a first microdrop and one or more second microdrops may be brought into contact with one another for a defined time sufficient so that, in particular owing to the interactions between the first microdrop and the second microdrop or microdrops, the second microdrop or microdrops undergo a change, for example a change of contents, and are then released to be analyzed. This may also allow the second microdrops to be changed for other second microdrops in the case of an error in the protocol prior to coalescence of the microdrops.
Third Microdrop
The method may comprise, after step (iii) or (iii′), the step (v) consisting of trapping a third microdrop in the second trapping zone or zones that no longer have a second microdrop, so that the first and the third microdrop are in contact with one another. The third microdrop may be identical to or different from the second microdrop. The third microdrop may be fused with the microdrop trapped in the first trapping zone or released, as described above for the second microdrop. Step (vi) may be repeated several times. This makes it possible for example to:
Release of the Capillary Trap
The method may comprise the step (vi) consisting of evacuating all of the microdrops present in the capillary trap outside the capillary trap, notably using a stream of fluid exerting an entraining force greater than the trapping forces that are exerted on the microdrops. Such a step may allow the microdrops to be released in order to analyze them.
The method may comprise the step consisting of taking a measurement of the state of the microfluidic system. This measurement may be performed before and/or after fusion and/or release of the drops.
Preferably, the final microdrop or microdrops obtained may comprise a means of identifying their contents, notably labeling by the presence of a number of beads or particles, by the presence of varied colors or shapes and/or by a colorimetric or fluorescence signal proportional to an initial concentration of a compound contained in one of the first microdrop and second microdrop.
The method described above may be carried out using the microfluidic system that is described hereunder.
Additional Steps
The method may comprise an additional step of
The step of observation or measurement may make it possible to determine the contents of each microdrop before and/or after fusion and for example determine the changes that occurred following fusion.
The observation step is, for example, particularly useful in the context of using a library of different microdrops for mapping the various microdrops before fusion.
II. Microfluidic Device
The invention also relates to a microfluidic device for trapping microdrops, notably for implementing the method as claimed in any one of the preceding claims, comprising a capillary trap having a first trapping zone and a second trapping zone arranged in such a way that a first microdrop trapped in the first trapping zone and a second microdrop trapped in the second trapping zone are in contact with one another in the capillary trap, the first and the second trapping zone being configured in such a way that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
The fact that the capillary trap has two zones having different trapping forces exerted on one of the microdrops makes it possible to have both selectivity of the trapped microdrops and trapping of the microdrops in a spatially predefined manner, notably to avoid the first microdrop occupying the second trapping zone, thus preventing the second microdrop being trapped in the latter. This is particularly true when a plurality of first and second microdrops are introduced into the microfluidic system.
The fact that the first and second microdrops are in contact allows them either to interact, or to be able to fuse easily.
Trapping Zones
The first and the second trapping zones are preferably cavities. The use of trapping zones in the form of a cavity facilitates manipulation of the microdrops and notably trapping them and/or releasing them.
The first and the second trapping zone may be separate.
As a variant, the first and the second trapping zone are joined together.
Preferably, the capillary trap lacks symmetry of revolution, when viewed from above. This anisotropy makes it possible to have trapping of the microdrops in a spatially predefined manner.
Preferably, the first trapping zone and the second trapping zone are arranged side by side, when viewed from above.
The first and the second trapping zones are preferably different by at least one of their dimensions. In particular, the first and the second trapping zones are of different heights, the first trapping zone notably being higher than the second trapping zone or the first and the second trapping zones are of different shapes, when viewed from above, the first trapping zone notably having a larger section than the second trapping zone. The difference in trapping force is then connected at least partially with the size, notably the height or the section when viewed from above, of the trapping zones.
“Height of the trapping zone” means, in cross section, the average height of the trapping zone of the microfluidic system.
The second trapping zone may become wider in at least one direction on approaching the first trapping zone. This makes it possible to guide the second microdrop in the direction of the first microdrop to keep it in contact with the latter. In fact, in order to minimize its surface energy, the second microdrop tends to move along the second trapping zone toward the zone with larger dimension.
The second trapping zone may become wider on approaching the first trapping zone, when viewed from above. Preferably, the divergence angle α is such that the second microdrop is always in contact with the two opposite walls defining it. The second trapping zone may become wider with a divergence angle α that is not zero, notably between 10° and 120°. The second trapping zone may have an approximately triangular or truncated triangular shape.
The second trapping zone may have a height that increases in the direction of the first trapping zone.
Preferably, the height of the second trapping zone is less than or equal to the largest dimension of the first trapping zone, better still less than or equal to half the largest dimension of the first trapping zone. The fact that the height of the second trapping zone is limited makes it possible to avoid the flow lines of the fluid being perturbed by the second trapping zone to the point of preventing the second microdrop 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 that has a high trapping force, in which the first microdrop is slightly deformed, in particular having a concave lower interface, which may facilitate, after sedimentation, bringing encapsulated elements into contact to form a cluster, for example of cells to form a spheroid.
Plurality of Second and/or First Trapping Zones
The capillary trap may comprise a plurality of second trapping zones arranged in such a way that each second trapped microdrop 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 in such a way that each first trapped microdrop is in contact with at least one of the second microdrop or microdrops or of the first microdrops trapped in the capillary trap.
Preferably, the first and second trapping zone(s) are arranged so that each second microdrop is joined to the or each first microdrop.
The first and second trapping zone(s) may be arranged in such a way that the second microdrops are all in contact with at least one first microdrop trapped in said capillary trap.
At least two second or first trapping zones may be configured so that the trapping forces that they would exert on one of said second liquid microdrops would be different. The second microdrops trapped by at least two second trapping zones may be different with respect to at least one of their properties, notably their largest dimension.
As a variant, all the second trapping zones of the capillary trap are identical.
Plurality of Capillary Traps
Preferably, the device comprises a plurality of capillary traps each comprising a first trapping zone and a second trapping zone, preferably arranged in such a way that the second microdrop trapped in the second trapping zone of the capillary trap is in contact with the first microdrop trapped in the first trapping zone of said capillary trap.
Each capillary trap may comprise one or more of the features described above.
All the capillary traps of the device may each comprise at least one first trapping zone and at least one second trapping zone.
As a variant, some of the capillary traps each comprise at least one first trapping zone and at least one second trapping zone and some of the capillary traps only comprise a single trapping zone, only allowing a single first microdrop to be trapped. These capillary traps comprising a single trapping zone may serve as a control during an experiment.
The device may comprise at least 10 capillary traps per square centimeter, better still at least 100 capillary traps per square centimeter. A large number of capillary traps notably makes it possible to do combinatorial chemistry, carry out screening of medicinal products, study protein crystallization, carry out titration of a chemical species, or personalize a treatment, notably in the case of cancer treatment.
At least two capillary traps may be different. For example, the device comprises a first capillary trap comprising n second trapping zones and a second capillary trap comprising p second trapping zones, n being different from p. Capillary traps of this kind can provide, after coalescence of the second microdrops with the first microdrops, microdrops trapped in the first trapping zones having different concentrations and/or sizes of drops. The microfluidic system may have more than two capillary traps having different quantities of second trapping zones in order to produce several concentrations and/or sizes of microdrops, notably a gradient of concentrations and/or sizes of microdrops. The microdrops obtained, with different concentrations, can form a panel of microdrops useful in the field of combinatorial chemistry, for studying protein crystallization, carrying out titration of a chemical species or personalizing a treatment, notably in the case of cancer.
As a variant, the capillary traps are all identical.
The device may comprise a channel having a trapping chamber, the capillary trap or traps being in the trapping chamber.
III. Second Aspect—Method of Manipulation
According to a second aspect, the invention also relates to a method of manipulating a plurality of first microdrops and a plurality of second microdrops in a microfluidic 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 trapping zone and a second trapping zone, said method comprising the steps consisting of:
the first and the second trapping zone of one and the same capillary trap being arranged in such a way that the first microdrop and the second microdrop are in contact with one another in the capillary trap, the capillary traps each having an anisotropic form.
The fact that there is a plurality of capillary traps makes it possible to form a plurality of pairs of first and second microdrops simultaneously. The different pairs of first and second microdrops may then be different or identical.
The fact that the capillary traps are anisotropic makes it possible to have predefined spatial positioning of the microdrops once they are trapped by the trapping zones.
Preferably, the first and the second trapping zones of each capillary trap are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the method or device according to the preceding aspects of the invention may apply to the method according to this aspect of the invention.
The method may be carried out using a microfluidic system for trapping microdrops 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 trapping zone and a second trapping zone arranged in such a way that a first microdrop trapped in the first trapping zone and a second microdrop trapped in the second trapping zone of the same capillary trap are in contact with one another, the capillary traps each having an anisotropic form.
Preferably, the first and the second trapping zones of each capillary trap are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the microfluidic system according to the preceding aspects of the invention may be applied to the microfluidic system according to this aspect of the invention.
IV. Third Aspect—Method of Manipulation
According to a third aspect, the invention also relates to a method for manipulating at least one first microdrop and at least one second microdrop in a microfluidic system comprising a capillary trap having a first trapping zone and a second trapping zone, the second trapping zone becoming wider in at least one dimension on approaching the first trapping zone, said method comprising the steps consisting of:
(i) trapping the first microdrop in the first trapping zone, and
(ii) trapping the second microdrop in the second trapping zone,
the first and second trapping zones of one and the same capillary trap being arranged in such a way that the first microdrop and the second microdrop are in contact with one another in the capillary trap.
The fact that the second trapping zone becomes wider in at least one dimension on approaching the first trapping zone makes it possible to guide the second microdrop toward the first microdrop during trapping thereof and keep the latter in contact with the first microdrop. In fact, in order to minimize its surface energy, the second microdrop tends to move along the second trapping zone toward the zone with larger dimension.
The second trapping zone preferably becomes wider, viewed from above, on approaching the first trapping zone.
The second trapping zone may become wider with a divergence angle α between 10° and 120°.
The second trapping zone may have a height that increases in the direction of the first trapping zone.
Preferably, the first and the second trapping zone are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the methods or devices according to the preceding aspects of the invention may apply to the method according to this aspect of the invention.
The method may be carried out using a microfluidic system for trapping microdrops comprising a capillary trap having a first trapping zone and a second trapping zone arranged in such a way that a first microdrop trapped in the first trapping zone and a second microdrop trapped in the second trapping zone of the same capillary trap are in contact with one another, the second trapping zone becoming wider in at least one dimension on approaching the first trapping zone.
Preferably, the first and the second trapping zone are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the microfluidic systems according to the preceding aspects of the invention may be applied to the microfluidic system according to this aspect of the invention.
V. Fourth Aspect—Method of Cellular Assembly
According to a fourth aspect, the invention also relates to a method of cellular assembly of at least one first microdrop containing first cells and of at least one second microdrop containing second cells, in a microfluidic system comprising a capillary trap having a first trapping zone and a second trapping zone, said method comprising the steps consisting of:
Such a method may allow microtissues to be created in vitro with a controlled architecture for very faithfully mimicking the conditions encountered in vivo. In fact, in the body, the different cell types are often arranged in tissues according to a specific architecture that it is important to reproduce optimally to recreate a function at the level of an organ. This three-dimensional culture with controlled architecture may be used with a view to transplantation in a patient. It is, for example, possible to culture glucagon-producing alpha cells, and insulin-producing beta cells, to create islets of Langerhans that may be transplanted into a patient's pancreas for treating diabetes. Similarly, hepatocytes and stellate cells may be combined in the context of a liver transplant.
Step (ii) may be carried out after aggregation of the first cells, in particular after formation of a first spheroid formed by adherence of the first cells to one another. If the first microdrop containing the first spheroid is liquid, the second cells will, after fusion of the two microdrops, be mixed with the contents of the first microdrop and then sediment to obtain the first spheroid directly. 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) may be carried out after aggregation of the second cells, in particular after formation of a second spheroid formed by adherence of the second cells together. Thus, the first and the second spheroid may be fused together.
The architecture of the microtissues obtained therefore depends on the experimental conditions.
The method may comprise an additional step of gelation of the first microdrops, said step taking place before step (iii) and preferably before step (ii). This makes it possible to compartmentalize the cells. In fact, if the first microdrops, which contain the spheroids, are gelled before arrival of the second microdrops, the second cells contained will no longer be able, after coalescence, to come directly into contact with the first spheroid, for example mammalian cells cannot pass through a matrix of agarose at 0.9 wt %. The first and the second cells can then only communicate with one another by the paracrine route.
The first and second cells may be of different cell types.
Preferably, the first and the second trapping zones are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the methods or devices according to the preceding aspects of the invention may apply to the method according to this aspect of the invention.
The method may be carried out using one of the microfluidic systems according to the preceding aspects.
VI. Fifth Aspect—Method of Cell Culture
According to a fifth aspect, the invention also relates to a method of cell culture in a microfluidic system of at least one first microdrop containing a cell culture and at least one second microdrop containing a culture medium, the microfluidic system comprising a capillary trap having a first trapping zone and a second trapping zone, said method of culture comprising the steps consisting of:
Sequential injection of the culture medium may make it possible to renew the latter several times in order for example to allow culture of the cell or cells in the first microdrop.
The second microdrop may comprise an active ingredient to be tested in order to model the intermittent nature of administration of a medicinal product. For example, a drop containing a spheroid of mammalian cells may be fused every 6 hours with a microdrop containing an active ingredient to be tested, notably a medicinal product.
The method may comprise, after step (iii), step (iv) consisting of repeating steps (ii) and (iii) to renew yet again the culture medium of the cell culture performed in the first microdrop.
Preferably, the first and the second trapping zone are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the methods or devices according to the preceding aspects of the invention may apply to the method according to this aspect of the invention.
The method may be carried out using one of the microfluidic systems according to the preceding aspects.
VII. Sixth Aspect—Method of Forming Gelled Microdrops
According to a sixth aspect, the invention also relates to a method of forming multilayer gelled microdrops of at least one first microdrop of a first gellable medium and of 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 zone and a second trapping zone, said method comprising the steps consisting of:
This makes it possible to form complex gel microdrops having variable shapes and/or mechanical properties, for example porosity and/or rigidity, and/or chemical properties, for example composition and/or concentration.
The method may comprise a step (v) taking place before or after step (iv) and consisting of gelling the second gellable medium.
When step (v) takes place after step (iv), 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. These microdrops of gels could be used with stem cells whose differentiation is notably controlled by the rigidity of the gel. Microdrops of gels with layers of different hydrogels containing different cell types may also make it possible to model the different layers of the skin in the context of cosmetic tests. A microdrop having a collagen core and an agarose outer layer with pores that are small enough may be used for creating a spheroid of neurons, only the axonal projections of which can be extracted via the pores of the outer layer.
When step (v) takes place before step (iv), the second gellable medium is gelled in the second trapping zone. This makes it possible to form complex gel microdrops having radially variable shapes, and/or mechanical and/or chemical properties. The microdrops formed then keep the shape and arrangement of the first and second microdrops before fusion. The disposition, shape and number of the different trapping zones therefore allow direct control of the shape of the final microdrop. Microdrops of this kind may make it possible to model complex shapes. The controlled shapes of the microdrops may also serve as an identifier of the latter.
Step (v) may take place before or after step (iii) of trapping in the second trapping zone.
Step (ii) may take place before or after step (i) of trapping in the first trapping zone.
The method preferably comprises a step (vi) consisting of repeating operations (iii) to (v).
The first and second gellable media may be different.
Preferably, the first and the second trapping zone are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the methods or devices according to the preceding aspects of the invention may apply to the method according to this aspect of the invention.
The method may be carried out using one of the microfluidic systems according to the preceding aspects.
VIII. Seventh Aspect—Method of Encapsulating the Cells
According to a seventh aspect, the invention also relates to a method for encapsulating at least one first microdrop and at least one second microdrop in a microfluidic system comprising a capillary trap having a first trapping zone and a second trapping zone, one of the first microdrop and second microdrop comprising a gellable medium and the other comprising a plurality of cells, said method comprising the steps consisting of:
This method notably makes it possible to obtain spheroids encapsulated in biological hydrogels. In fact, in order to be able to form spheroids in microdrops in a controlled manner, it must be possible to keep the contents of the drop liquid during the time of formation of the spheroids. Agarose is very suitable for this protocol as it is a heat-sensitive hydrogel. It remains liquid at 37° C. and then solidifies after 30 min at 4° C. and remains solidified after returning to 37° C. However, mammalian cells cannot adhere to agarose and they cannot digest it either. This matrix is therefore very different from the extracellular matrix encountered in the body. The use of hydrogels such as for example type I collagen, fibronectin, Matrigel® or gelatin might be preferable for better simulation of natural conditions. However, it is more difficult to control their gelation. For example, type I collagen cannot be kept liquid for a long time with favorable conditions for cell culture (low temperature or acid pH). If cells are encapsulated in a drop of collagen that is gelled rapidly after trapping the cells, rather than adhere to one another and form a spheroid, the cells will adhere to the collagen and migrate individually along its fibers.
This problem may be solved by the aforementioned method. In fact, the cells may be encapsulated in the first liquid microdrops within the first trapping zone so as to form a spheroid. Second microdrops may then be supplied, which will lodge in the second trapping zone and which contain one of the biological hydrogels mentioned above, notably in high concentration. Once these second microdrops are trapped, the first and second microdrops in contact are fused immediately and the biological hydrogel, still liquid, will mix with the first microdrop that contains the spheroid. Gelation may then take place and will therefore encapsulate the spheroid in an extracellular matrix representative of the biological conditions encountered in vivo.
Step (ii) may be carried out after aggregation of the first cells, in particular after formation of a spheroid formed by adherence of the first cells to one another.
Preferably, the first and the second trapping zone are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the methods or devices according to the preceding aspects of the invention may apply to the method according to this aspect of the invention.
The method may be carried out using one of the microfluidic systems according to the preceding aspects.
IX. Eighth Aspect—Method of Dilution
According to an eighth aspect, the invention also relates to 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 comprising a first trapping zone and p second trapping zones, n being different from p, said method comprising the steps consisting of:
the first and the second trapping zones of one and the same capillary trap being arranged in such a way that each second microdrop is in contact with at least one of the first or second microdrops of the same first or second capillary trap and so that, in each of the first and second capillary trap, at least one of the second microdrops is in contact with the first microdrop of the same first or second capillary trap, and then
If the first drops contain a compound of interest at constant concentration, a spatial gradient of concentration may be obtained after coalescence with the second microdrops, which may for example contain a diluent. With such a method it may be possible to obtain a panel of microdrops with different controlled concentrations, starting from microdrops with the same concentration. This may for example be advantageous for forming a panel of microdrops of different concentrations for application in combinatorial chemistry, in the investigation of protein crystallization, in a subsequent method for titration of a chemical species, or in the personalization of a treatment, notably in the case of cancer.
Preferably, the first and the second trapping zones of each capillary trap are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the methods or devices according to the preceding aspects of the invention may apply to the method according to this aspect of the invention.
The method may be used by the microfluidic system for dilution of microdrops comprising a first capillary trap comprising a first trapping zone and n second trapping zones and a second capillary trap comprising a first trapping zone and p second trapping zones, n being different from p, the first and second capillary trap being configured so that the second microdrops trapped in each of the second trapping zones are in contact with the first microdrop trapped in the corresponding first trapping zone in the first and the second capillary trap.
Preferably, the first and the second trapping zones of each capillary trap are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the microfluidic systems according to the preceding aspects of the invention may be applied to the microfluidic system according to this aspect of the invention.
X. Ninth Aspect—Method of Screening
According to a ninth aspect, the invention also relates to a method of screening a plurality of first microdrops with a plurality of second microdrops in a microfluidic system comprising a plurality of capillary traps, each capillary trap having a first trapping zone and a second trapping zone, the first microdrops forming a first panel of microdrops that are identical or of which at least y are different and the second microdrops forming a second panel of microdrops of which at least z are different, said method comprising the steps consisting of:
Such a method allows rapid screening of a large number of reaction conditions in a single microfluidic system.
The fact that the microdrops are static during the reaction makes it easier to obtain kinetic data. There is also the advantage of economy of compounds, through the use of very small volumes in the microdrops.
The first panel of microdrops may comprise microdrops that are different at least in their contents, notably their concentration of a first compound of interest.
The second panel of microdrops may comprise microdrops that are different at least in their contents, notably their concentration of a second compound of interest.
The first or second microdrops may 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 we wish to optimize. Thus, it will be possible to perform several reactions in parallel on a small volume in order to determine the initial concentrations of compounds giving the best results.
Preferably, the method comprises an additional step (iv) of observation or of measurement, notably by imaging, by colorimetric, fluorescence, spectroscopic (UV, Raman) or temperature measurement, before step (iii). Such a step makes it possible to map the disposition of the different microdrops.
Preferably, the method comprises an additional step (v) of observation or of measurement, notably by imaging, by colorimetric, fluorescence, spectroscopic (UV, Raman) or temperature measurement, after step (iii).
As a variant, the first panel of microdrops comprises proteins, the first microdrops being identical, and the second panel of microdrops comprises different concentrations of a solution allowing protein crystallization, notably a saline solution. The method may then make it possible to study protein crystallization as a function of the concentration of crystallization solution. In fact, the optimum crystallization conditions vary from one protein to another.
As a further variant, the first panel of microdrops comprises a compound, the first microdrops being identical and the second panel of microdrops comprises a titrating species at different concentrations. This application may be particularly advantageous in the case of assay of reagents that are expensive or available in small amounts.
As a further variant, the first panel of microdrops comprises one or more cells and the second microdrops each comprise a medicinal product to be screened at a defined concentration.
In a similar configuration, liver cells are cultured in the form of spheroids in the first microdrops, and a second microdrop containing a medicinal product at different concentrations, whose toxicity we wish to evaluate, is supplied in each of the second trapping zones.
By analyzing the results of viability a few days after coalescence of the microdrops it is possible to determine the concentration that kills half of the cellular population.
This method may also make it possible to evaluate the interactions between different antibiotics. It is possible to create microdrops forming a panel of microdrops having different concentrations of antibiotics A and B and fuse them with microdrops containing bacteria. The microdrops containing the bacteria may form a panel of microdrops with different concentrations of 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.
By using microfluidics it is in addition possible to use very small volumes, which may be very advantageous in the context of rare samples such as cells obtained from a biopsy. The system may 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 from a patient who has undergone a biopsy and subject them to various active substances at multiple concentrations by supplying second microdrops. After fusion of the pairs of microdrops containing cells and active substances, it is possible to determine which active substance will be the most effective, and at what concentration, for a particular patient, using just a single chip and a minimum number of cells obtained from the biopsy.
Preferably, the first and the second trapping zones of one and the same capillary trap are configured so that the trapping forces that would be exerted by the first and by the second trapping zone on a same first or second liquid microdrop would be different.
One or more of the features described above in connection with the methods or devices according to the preceding aspects of the invention may apply to the method according to this aspect of the invention.
The method may be carried out using one of the microfluidic systems according to the preceding aspects.
The invention may be better understood on reading the following description of nonlimiting embodiment examples of the invention, referring to the appended drawing, in which:
The invention relates to a method for manipulating at least one first and one second microdrop in a microfluidic system.
The microfluidic system 5 comprises an upper wall 7 and a lower wall 8, between them forming a channel 9 for circulation of the microdrops and at least one capillary trap 12.
Capillary Trap
In the example illustrated in
The first and the second trapping zones 15 and 18 exert different trapping forces on a given microdrop, in particular owing to their difference in shape. Here, the first trapping zone 15 exerts a larger trapping force than the second trapping zone 18.
When a first microdrop 20 is introduced into the microfluidic system it is trapped in the trapping zone having the largest trapping force for this microdrop, in this case the first trapping zone 15. When the second microdrop 25 is introduced it is trapped in the free trapping zone, here the second trapping zone 18, as is illustrated in
In the figures, the first microdrops 20 are shown in black and the second microdrops 25 are shown transparent, but without this representing a particular difference in contents between the two microdrops.
The first trapping zone 15 has a diameter a approximately equal to the apparent diameter D1, viewed from above, of the first microdrop 20, once trapped in the first trapping zone.
The two trapped microdrops 20 and 25 are in contact with one another notably on account of the small distance between the two trapping zones 15 and 18 relative to the diameters of the two microdrops 20 and 25. Moreover, the two microdrops 20 and 25 are kept in contact owing to the triangular shape of the second trapping zone 18. In fact, the second microdrop 25, being in contact with two opposite walls 27 and 28 of the second trapping zone 18 moving away from one another in the direction of the first trapping zone 15, is caused, through its natural tendency to always minimize its surface energy, to move in translation between the two opposite walls 27 and 28 in the direction of widening of the walls 27 and 28, i.e. toward the first trapping zone 15 and therefore the first microdrop 20.
In
In the example illustrated in
The second trapping zone 18 exerts a trapping force on the second microdrop 25 that is greater than what it would exert on the first microdrop 20. In fact, although the design of the second trapping zone is the same regardless of the diameter of the microdrop, the diameter of the second microdrop 25 adapts better to the shape and size of the second trapping zone 18 than of the first microdrop 20. However, it may be otherwise, and the second microdrop 25 may be of the same diameter as the first microdrop 20.
As a variant, the first microdrop and the second microdrop 20 and 25 are different from one another with respect to another of their properties, notably their surface state, their viscosity or their weight.
As a variant illustrated in
As a variant, illustrated in
As a variant illustrated in
As a variant, not illustrated, the two second trapping zones 18a and 18b may exert identical forces on the second trapped microdrops 25a and 25b.
As a variant, not illustrated, the capillary trap may have more than two second trapping zones configured to form a string of trapped second microdrops, itself in contact, via at least one of the second microdrops forming it, with the first microdrop trapped in the first trapping zone.
It is thus possible to envisage more complex shapes of capillary traps comprising a plurality of trapping zones configured so that the trapped microdrops are all joined together directly or via other microdrops.
As a variant illustrated in
The second trapping zones 18 may be uniformly distributed around the first trapping zone 15, as is illustrated. However, it may be otherwise.
As a variant illustrated in
As a variant illustrated in
The invention is not limited to the examples of shape of the capillary trap 12 described above. The capillary trap 12 may have various shapes, notably as a function of the required application.
For example, as illustrated in
As a variant illustrated in
As a variant illustrated in
As illustrated in
As illustrated in
As illustrated in
As illustrated in
The first trapping zone 15 may be of square shape, as illustrated in
As a variant, the second trapping zone 18 is of triangular shape but joined to the first trapping zone 15 by one of its corners, as illustrated in
As a further variant, the second trapping zone 18 is of polygonal shape, notably hexagonal, as illustrated in
As a further variant, the capillary trap 12 may comprise two second trapping zones 18 fused to the first trapping zone 15 at opposite corners of the latter, as illustrated in
As illustrated in
As a variant illustrated in
As illustrated in
The first trapping zone 15 may be of rectangular shape and the second trapping zone 18 of square shape fused to the first trapping zone 15 by a short side of the rectangle. Depending on the size of the first microdrops 20, the first trapping zone 15 may then trap a single first microdrop 20, as illustrated in
As a variant illustrated in
The first trapping zone 15 may be of oval shape and the second trapping zone 18 may be fused to the latter starting from its long side, as illustrated in
The capillary trap may comprise a plurality of second trapping zones 18, at least two of which are different, notably by their sizes, as is illustrated in
In the example illustrated in
As a variant illustrated in
In the example illustrated in
In the examples illustrated in
In the examples illustrated in
In cross section, the first trapping zone 15 and the second trapping zone 18 may be of constant height over their entire width.
As a variant illustrated in
As a further variant illustrated in
As a further variant illustrated in
As a variant illustrated in
As a variant, the capillary trap 12 is formed at least partially by a cavity of one of the side walls of the microfluidic system.
As a variant illustrated in
As a variant illustrated in
Microfluidic Device
The channel 9 for circulation of the microdrops may comprise a plurality of capillary traps 12.
In particular, channel 9 may comprise a two-dimensional trapping chamber 30 in which the capillary traps 12 are distributed spatially according to two spatial directions in a table or matrix, as illustrated in
The number of capillary traps 12 in the trapping chamber 30 may range from a single one per chamber to several thousand per cm2.
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 largest microdrops intended to be trapped, notably greater than or equal to the apparent diameter, viewed from above, of a first drop confined in the channel between the walls 7 and 8 outside of the capillary traps, for example between 20 μm and 1 cm.
The number of capillary traps may be greater than or equal to 200 capillary traps per cm2, better still 2000 capillary traps per cm2. Thus, controlled combination of microdrops may be performed in hundreds, or even tens of thousands of traps in parallel in the same trapping chamber 30.
The capillary traps 12 may be as described above.
The capillary traps 12 may all be identical.
As a variant, at least two capillary traps 12 may be different, notably by their shapes, sizes, heights or orientations, or by the number, shape, height or orientation of the first and second trapping zones 15 and 18. This makes it possible to have different conditions as a function of the capillary trap 12.
As a variant that is not illustrated, channel 9 may be one-dimensional and comprise an array of capillary traps 12 distributed along its length.
The invention is not limited to the shapes of microfluidic system described above. The microfluidic system may have different shapes, notably as a function of the required application.
Microdrops
Preferably, channel 9 is filled with a fluid in which the microdrops are immiscible. This fluid may be stationary or moving. When the latter is moving, the stream of fluid is preferably oriented along the lines of fluid circulation (not shown) and circulates from a fluid inlet 31 to a fluid outlet 32.
The microdrops are, for example, aqueous microdrops in an oily liquid or microdrops of oil in an aqueous liquid.
Preferably, the first and second microdrops 20 and 25 have diameters D1 and D2 of the order of a micrometer, notably between 20 and 5000 μm.
The first microdrops 20 are preferably different from the second microdrops 25, notably with respect to their sizes and/or their compositions.
The first microdrops 20, or the second microdrops 25, may form a panel of microdrops, at least a certain number of which are different.
The first and/or the second microdrops 20 and 25 may comprise an identifying compound allowing them to be identified before, during and/or after coalescence of the first and second microdrops 20 and 25 in contact. This or these identifying compound(s) may be for example a certain number of beads or particles, compounds of varied colors or shapes or compounds emitting a colorimetric or fluorescence signal proportional to their concentration in the microdrop. In the case of 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 map the microdrops trapped in the trapping chamber.
As a variant, the identifying compound or compounds of the first microdrops interact with one or more identifying compounds of the second microdrops so as to allow identification of the microdrop obtained after fusion. For example, fusion of the microdrops may lead to a chemical reaction, at least one of the products of which may be identified.
Preferably, channel 9 is filled with a fluid containing a surfactant. The latter allows stabilization of the microdrops and reproducibility of their formation. The surfactants in addition make it possible to prevent spontaneous coalescence of the microdrops in the case of contact while they are conveyed from the production device to the capillary traps or in the capillary traps.
For aqueous microdrops, the surfactant is for example a compound selected from PEG-di-Krytox in a fluorinated oil or SPAN®80 in a mineral oil.
For microdrops of oil, the surfactant is for example sodium dodecyl sulfate.
As a variant, the microdrops are stabilized by some other means, notably the microdrops may be gelled, or stabilized by adsorption of amphiphilic nanoparticles as described in the article of Pan, M., Rosenfeld, L., Kim, M., Xu, M., Lin, E., Derda, R., & Tang, S. K. Y. (2014). Fluorinated Pickering Emulsions Impede Interfacial Transport and Form Rigid Interface for the Growth of Anchorage-Dependent Cells. Applied Materials & Interfaces, 6, 21446-21453, incorporated here by reference.
Method of Manipulation
An example of a method of manipulating the first and second microdrops is illustrated in
The first microdrops 20 are produced in step 40. Many methods have already been proposed for forming these first microdrops in a mobile phase. For example, the following examples of methods may be mentioned:
These methods notably make it possible to form a plurality of microdrops of approximately equal dimensions. The dimensions of the microdrops obtained may be controlled by modifying the parameters of formation of the microdrops, notably the velocity of circulation of the fluids in the device and/or the shape of the device.
The first microdrops 20 may be produced on the same microfluidic system as the method or on a different device. In the latter case, the first microdrops 20 may 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.
After formation of these first microdrops 20, the latter may be conveyed to the capillary trap 12 by entrainment by a stream of a fluid and/or by slopes or reliefs in the form of rails of the channel 9. In both cases, the addition of rails may make it possible to optimize the filling of the capillary trap 12, selectively, for example in combination with the use of an infrared laser, as described by E. Fradet, C. McDougal, P. Abbyad, R. Dangla, D. McGloin, and C. N. 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, December 2011.
If production of the microdrops is carried out outside the microfluidic system, they may be transported from storage to the microfluidic system directly via a tube connecting for example the production system and the trapping system or by aspiration and injection with a syringe.
The first microdrops 20 are entrained in the microfluidic system in such a way that the entraining force to which they are subjected is less than the trapping force of the first trapping zones 15 on the first microdrops 20. The first microdrops 20 are then trapped, in step 42, in the capillary trap 12, in particular in the first trapping zones 15. If the entraining stream exerts on the first microdrops 20 an entraining force on the first microdrops 20 that is greater than the trapping force of the second trapping zones 18, the latter are not trapped in the second trapping zones 18 that remain free.
Otherwise the first microdrops 20 may be trapped in the second trapping zones 18, especially if the sizes of the traps and of the drops are suitable. It is then possible to remove them from the latter by increasing the entraining force applied to all of the first microdrops 20, for example by increasing the velocity of the stream of fluid or, when there is none, by adding a stream of fluid in a step 44.
As a variant, the first microdrops 20 may be formed by a method called “breaking of the drops in capillary traps” described for example in international application WO 2016/059302, the contents of which are incorporated here by reference. In this case, the first microdrops 20 are formed directly in the first trapping zones 15.
The second microdrops 25 are produced in step 46. This step is shown after step 44 but it could take place beforehand. 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 microdrops 25 are entrained in the microfluidic system in such a way that the entraining force to which they are subjected is less than the trapping force of the second trapping zones 18 on the second microdrops 25. The second microdrops 25 are then trapped, in step 48, in the second trapping zones 18. When the second microdrops 25 are entrained by a stream of fluid, the latter exerts an entraining force on the first microdrops 20 trapped in the first trapping zones 15 that is preferably less than or equal to the trapping force of the first trapping zones 15 on the first microdrops 20 so that the latter remain trapped.
At each stage, the method may comprise a step of first measurement of the state of the system. This measurement may be simple imaging, or for example colorimetric, fluorescence, spectroscopic (UV, Raman) or temperature measurement. This measurement may be particularly useful in the context of using panels of different first and/or second microdrops 20 comprising an identifying compound as described above.
When several microdrops are in contact in one and the same trap, it is possible to fuse them in a controlled manner to mix their contents in step 50. This coalescence may or may not be selective.
To fuse all of the microdrops in contact in the microfluidic system, notably in the trapping chamber 30, the latter is perfused with a surfactant-free fluid. The concentration of surfactant in the fluid of the microfluidic system decreases, which makes it possible to shift the equilibrium of adsorption of surfactant at the interface toward desorption. The microdrops lose their stabilizing effect and fuse spontaneously with the microdrops with which they are in contact.
As a variant, 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 microdrops.
As a further variant, all of the microdrops in contact in the microfluidic system, notably in the trapping chamber 30, are fused by applying an external physical stimulus, such as mechanical waves, pressure waves, a temperature change or an electric field. As is illustrated in
To fuse the microdrops selectively, an infrared laser may be used, as described by E. Fradet, P. Abbyad, M. H. Vos, and C. N. Baroud, in “Parallel measurements of reaction kinetics using ultralow volumes,” Lab Chip, Vol. 13, No. 22, pp. 4326-30, October 2013, or localized electrodes 37 at the level of the interfaces of microdrops between the trapping zones may be activated, as illustrated in
The invention is not limited to the examples of coalescence described above. Any method making it possible to destabilize the interface between two microdrops in contact may be used for fusing the microdrops.
It is then possible to measure the state of the microdrops obtained and/or observe the latter in real time. This makes it possible, for example, to study the kinetics of chemical or biochemical reactions.
Selective Trapping
When the capillary trap or traps 12 have a plurality of second trapping zones 18, the position of the second trapping zone or zones 18 relative to the direction and the sense of the entraining force being exerted on the second microdrops 25 may allow selective trapping to be carried out.
Take the case of a capillary trap with two second trapping zones 18m and 18v arranged on either side of a first trapping zone 15 and aligned in the direction of the entraining force exerted, as illustrated in
As is illustrated in
Example of Chronology of Microdrop Trapping
In an embodiment example illustrated in
Selective Trapping of the Two Microdrops is Thus Obtained, in the Two Zones of the Capillary Trap Respectively.
Trapping According to the Size of the Microdrops
The second trapping zones 18 of the various capillary traps 12 in one and the same microfluidic system may have different properties, notably different sizes. This makes it possible, for example, to trap different second microdrops 25 in the different capillary traps 12 in order to obtain different microdrops. For example, for a given concentration of an element included in the second microdrops, the amount 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 trapping chamber 30, it is possible to trap second microdrops 25 of different sizes selectively, with a size of second microdrop 25 corresponding to each size of second trapping zone 18.
It is preferable to place the capillary traps 12a, 12b and 12c by trapping forces of the second trapping zones 18a, 18b and 18c relative to the largest of the second microdrops 18a increasing in the direction of the entraining force of the second microdrops, notably in the direction of the stream of fluid, in the microfluidic system. Thus, the second microdrops 25a, 25b and 25c first encounter the second trapping zones 18c, in which only the smallest second microdrops 25c are trapped, then the second trapping zones 18b in which only the second microdrops 25b are trapped and finally the second trapping zones 18a in which the largest second microdrops 25a are trapped.
As a variant, the microdrops 25a, 25b and 25c differ by another of their parameters, and they notably comprise different elements.
This makes it possible to obtain a panel of microdrops, at least some of which are different, notably in respect of their concentration of a compound and/or their composition.
Sequential Coalescence
Steps 46, 48 and 50 may be repeated, as is illustrated in
Note that if the coalescence step consists of removing the surfactant from the external phase or of destabilizing it chemically, a stabilization step may be necessary between the different coalescences. In practice, it is necessary to perfuse the trapping chamber with a fluid that is immiscible with the microdrops containing surfactant before bringing one or more new microdrops into the capillary trap.
The ability to perform sequential coalescence has several applications. If a first microdrop 20 trapped in a first trapping zone 15 of a capillary trap 12 contains cells (for example bacteria, yeasts or mammalian cells), sequential coalescence may allow the culture medium to be renewed several times by sequentially fusing second microdrops 25 containing a culture medium at predetermined times.
This may also serve for modeling the intermittent nature of administration of a medicinal product. For example, 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 microdrop 25 of medicinal product.
Drop Concentration Gradient
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.
As is illustrated in
Panels of Microdrops
Injection of panels of microdrops comprising different compounds and/or different concentrations in a chamber containing capillary traps 12 as described above offers many applications.
Take the example of a trapping chamber of 2 cm2 having 1000 capillary traps each making it possible to trap a first and a second microdrop 20 and 25. 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. By fusing the first and the second microdrops 20 and 25 in the capillary traps 12, it is possible to obtain a matrix of microdrops each corresponding statistically to one combination among the 200 possible combinations of first and second microdrops 20 and 25.
The fact that the microdrops are static also makes it easier to obtain kinetic data. There is also the advantage of economy of reagents, through the use of very small volumes in the microdrops.
The trapping chamber 30 may have a surface area greater than 2 cm2, giving a further large increase in the number of different reactions that may be carried out in parallel in a microfluidic system.
The compounds contained in the first and second microdrops 20 and 25 may be chemical molecules that will react with one another and whose initial concentrations are required to be optimized. The method described above makes it possible to carry out large-scale combinatorial chemistry. The microdrops may then comprise one or more identifying means. This may make it possible for example to measure the concentration of the end product, for example by fluorescence or spectroscopy.
As a variant, the first and/or second microdrops 20 and/or 25 may contain proteins, enzymes, and cells at various concentrations.
The microfluidic system and the method described above may make it possible to investigate protein crystallization. In fact, 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. However, the protein is always available in very small amounts and the optimum crystallization conditions vary from one protein to another.
For example, using a trapping chamber 30 with several capillary traps 12, each making it possible to trap a first and a second microdrop 20 and 25, gives a first microdrop panel comprising a saline solution in different concentrations and a second microdrop panel comprising a protein of interest in different concentrations. By fusing the first and the second microdrops 20 and 25 in the capillary traps 12, it is possible to obtain a matrix of microdrops representing conditions of different concentrations of saline solution and proteins, so as to be able to determine the concentrations allowing optimum protein crystallization.
By immobilizing, on the capillary traps 12, first microdrops 20 comprising an element of interest in identical concentration in the whole trapping chamber and by fusing the latter with second microdrops 25 obtained from a panel of microdrops comprising a titrating species in different concentrations, it is possible to perform titration of the species of interest contained in the first microdrops 20. This application may be particularly advantageous in the case of reagents that are expensive or available in small amounts.
The method presented here may also be very useful for screening medicinal products. For example, cancer cells may be cultured, in individualized form or in the form of spheroids, in first microdrops 20 trapped in each of the first trapping zone 15 of the capillary trap 12, and after culture for some days, it is possible to coalesce them in each trap with a second microdrop 25 containing a medicinal product to be screened, the second microdrops 25 being obtained from a panel of microdrops containing different medicinal products.
In a similar configuration, we may imagine the culture of liver cells in the form of spheroids in first trapped microdrops 20 and the supply, to each of the capillary traps 12, of a second microdrop 25 containing a medicinal product, whose toxicity we wish to evaluate, each microdrop being obtained from a panel of microdrops comprising the medicinal product in different concentrations. By analyzing the results for viability some days after coalescence, it is possible for example to deduce the concentration of this medicinal product that kills half of the cellular population.
This method may also allow evaluation of the interactions between different antibiotics. It is possible to create a panel of second microdrops 25 containing one or more antibiotics in different concentrations and fuse them in the trapping chamber 30 with first microdrops 20 containing bacteria. The first microdrops 20 may comprise a bacterium in different concentrations. This makes it possible to explore a space with 3 parameters.
The application of microfluidics may be very advantageous in the context of rare samples such as biopsies. The microfluidic system may 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 first trapped microdrops 20, tumor cells from a patient who has undergone a biopsy and subject them to different medicinal products at multiple concentrations by feeding the second microdrops 25 into the trapping chamber 30. After coalescence of the pairs of microdrops with cells and the medicinal product, the most effective medicinal product and its concentration for a particular patient may be determined using only a single trapping chamber 30 and a minimal number of cells obtained from the biopsy.
Tissue Engineering
The method as described above may make it possible to fuse microdrops containing cells, which may or may not be of different cell types, for accurately forming microtissues.
The capillary trap may be as illustrated in
A first microdrop 20 may contain cells of a first cell type in a liquid medium and may be trapped in the first trapping zone 15 of said capillary trap 12 to form, after immobilization for one day, a first spheroid spontaneously by sedimentation of the cells. Preferably, the microfluidic system does not have a stream of fluid near the capillary trap during formation of the first spheroid so that the liquid of the first microdrop 20 is not caused to move. A second microdrop 25 containing cells of a second cell type in a liquid medium may be trapped in the second trapping zone 18. After coalescence of the first and of the second microdrop, a culture of cells of two different types is obtained, the architecture of which depends in particular on the experimental conditions.
If the first microdrop 20 is liquid, the cells of the second microdrop 25 mix, after coalescence, with the contents of the first microdrop 20 and then settle, giving the spheroid of cells of the first cell type directly.
If the cells of the second microdrop 25 have had time before coalescence to form a second spheroid, coalescence of the two microdrops 20 and 25 leads to fusion of the first and second spheroids.
If coalescence takes place before the cells of the second microdrop 25 have had time to form a spheroid, the cells will be deposited after sedimentation on the surface of the first spheroid.
If now one of the two microdrops 20 or 25 is gelled before the coalescence operation, the two cellular populations are compartmentalized. In fact, if the first microdrop 20, which contains the first spheroid, is gelled before the second microdrop 25 arrives, the cells of second cell types will no longer be able, after coalescence of the microdrops 20 and 25, to come directly into contact with the first spheroid owing to the presence of the gel. For example, mammalian cells cannot pass through a matrix of agarose at 0.9 wt %. The two groups of cells can then only communicate with one another by the paracrine route.
The example given here is for a capillary trap 12 trapping a first and a microdrop 20 and 25 but it is possible to obtain more complex architectures of microtissues with a capillary trap 12 allowing more than two microdrops to be trapped and/or methods as described above consisting of coalescing several microdrops sequentially by varying or not varying the orientation of the stream of fluid. It is also possible to use a plurality of capillary traps as described above to form a plurality of microtissues in parallel.
This technique for forming microtissues may make it possible to create microtissues in vitro with a controlled architecture for very faithfully mimicking the conditions encountered in vivo. In fact, in the body the different cell types are often arranged in tissues according to a specific architecture that is important for recreating a function at the level of an organ.
The latter may also be used for the purpose of transplantation in a patient. For example, glucagon-producing alpha cells and insulin-producing beta cells may be combined to create islets of Langerhans intended to be transplanted into a patient's pancreas to treat diabetes. Similarly, hepatocytes and stellate cells could be combined in the context of liver transplant.
Hydrogels
The method as described above may also be used for creating multilayer gel microdrops.
The capillary trap 12 may be as described above and may comprise a first trapping zone 15 and a second trapping zone 18.
As is illustrated in
As a variant illustrated in
The method may also make it possible to obtain spheroids encapsulated in biological hydrogels.
In order to be able to form spheroids in microdrops in a controlled manner it is necessary to be able to keep the contents of the microdrop liquid during the time of formation of the spheroid. Agarose is very suitable for this protocol as it is a heat-sensitive hydrogel. It remains liquid at 37° C. (ultra low gelling agarose) and then solidifies after 30 min at 4° C. and remains solidified after returning to 37° C. However, mammalian cells cannot adhere to agarose and they cannot digest it either. This matrix is therefore very different from the extracellular matrix encountered in the body. The use of hydrogels such as for example type I collagen, fibronectin, Matrigel® or gelatin could be preferable for better simulation of natural conditions. However, it is more difficult to control their gelation. For example, type I collagen cannot be kept liquid for a long time with favorable conditions for cell culture (low temperature or acid pH). If cells are encapsulated in a collagen microdrop that is gelled quickly after trapping, rather than adhering to one another and forming a spheroid, the cells will adhere to the collagen and migrate individually along its fibers.
This problem can be solved by the method according to the invention.
The capillary trap may be as illustrated in
A first microdrop 20 may contain cells of a first cell type in a liquid medium and may be trapped in the first trapping zone 15 of said capillary trap 12 to form, after immobilization for one day, a spheroid spontaneously by sedimentation of the cells. Preferably, the microfluidic system does not have a stream of fluid near the capillary trap during formation of the spheroid, so that the liquid of the first microdrop 20 is not caused to move. A second microdrop 25 containing one of the biological hydrogels mentioned above in high concentration may be trapped in the second trapping zone 18. Once this second microdrop is trapped, the two microdrops are fused immediately in such a way that the biological hydrogel, still liquid, mixes with the first microdrop that contains the spheroid. Gelation then takes place and the spheroid is encapsulated in an extracellular matrix representative of the biological conditions encountered in vivo.
This technique of spheroid encapsulation may be combined with the technique for forming microtissues described above to allow more complex architectures of microtissues to be obtained.
The following nonlimiting examples describe embodiment examples of the invention as described above.
An experiment was carried out to demonstrate the feasibility of the method. The trapping chamber 30 used is of 2 cm2 and contains 393 identical capillary traps similar to that in
a=250 μm,
b=c=150 μm,
H=100 μm, and
h=50 μm.
The capillary traps 12 are distributed according to a matrix as illustrated in
The microdrops comprise food dyes. Drops of 1 μL with five different colors ranging from blue to green to yellow were formed by the “micro-segmented flows” technique. These 1 μL drops were fractionated into many first monodisperse nanoliter microdrops 20 using a slope (method described at point f) above). These first microdrops 20 of various colors were then mixed to form a first panel of microdrops comprising different colors and were then injected into the trapping chamber 30 containing the capillary traps 12. The size of the first microdrops 20 was adjusted so that they fully occupy the first trapping zones 15. The second trapping zones 18 remain empty.
In the same way, five hues of 1 μL drops ranging from transparent, colorless to red were formed and then fractionated into second microdrops smaller than the first microdrops owing to a differently designed slope. These second microdrops were mixed to form a second panel of microdrops comprising different colors and were then injected into the microfluidic chamber, where they are trapped in the second free trapping zones 25.
A matrix of pairs of first and second microdrops 20 and 25 as illustrated in
This experiment demonstrates that it is possible to combine different reagents in different concentrations within one and the same trap, in parallel in a trapping chamber 30, after coalescence of pairs of different compositions.
An experiment was carried out for obtaining spheroids resulting from two successive fusions of separate spheroids.
The microfluidic system comprises a trapping chamber 30 having a matrix of capillary traps as illustrated in
H=165 μm, and
h1=388 μm,
h2=80 μm,
c=200 μm,
a=400 μm.
Rat hepatic cells (H4IIEC3) were first encapsulated in first microdrops 20. The first microdrops 20 were trapped in the first trapping zones 15 and, after sedimentation, the cells collect at the bottom of each drop to form a first spheroid 130. After one day, necessary for formation of these first spheroids, second microdrops 25 were trapped in the second trapping zones 18, as can be seen in
After coalescence in each trap, the second spheroid 135 sediments and comes into contact at the bottom of the first microdrop with the first spheroid 130 in a new first microdrop 140, as can be seen in
This operation was then repeated, this time with third microdrops 85 encapsulating H4IIEC3 cells that had been colored green (CellTracker Green®), as can be seen in
Therefore we finally obtain a matrix of three-colored spheroids, as illustrated in
This experiment demonstrates the potential of the technique for applications connected with tissue engineering. In fact, rather than fusing spheroids of one and the same cell type but with different colors, we may easily imagine fusing spheroids of complementary cell types to create functional microtissues with a well-defined architecture.
An experiment was conducted to determine, in a single microfluidic system, the concentration beyond which a medicinal product (acetaminophen) becomes toxic to liver cells (rat hepatoma, H4IIEC3 cells).
The microfluidic chamber used containing 252 identical traps on 2 cm2 in
The first microdrops 20 of agarose that is liquid at 37° C. (ultra-low gelling), at 0.9 wt % diluted in culture medium containing H4IIEC3 cells, were trapped in the first trapping zones 15, the second trapping zone 18 remaining free. The first microdrops 20 were then cultured for one day to allow the cells to adhere to one another to form a spheroid by first microdrops 20. The first microdrops 20 were then gelled by application of a temperature of 4° C. for 30 min.
In parallel, acetaminophen was dissolved at high concentration in culture medium. Fluorescein at high concentration was added to this solution. The solution obtained was diluted to different concentrations with pure culture medium to form 14, drops at different concentrations. These drops were then fractionated into second microdrops 25 by means of a slope and then mixed before being injected into the trapping chamber 30 containing the first microdrops 20. These second microdrops 25 were smaller than the first microdrops 20 and were trapped in the second trapping zones 18.
By taking a fluorescence image before fusion, the different levels of fluorescence that are correlated with the concentration of medicinal product in the second microdrops 25 could be identified. Even if these drops were immobilized randomly in traps, it is then possible to find the concentration of acetaminophen with which it was combined in the capillary trap 12, corresponding to each spheroid.
The chamber was perfused with HFE-7500 (fluorinated oil) containing 1H,1H,2H,2H-perfluorooctan-1-ol at a concentration of 20 vol % in order to fuse the microdrops in contact. The acetaminophen then diffused through the gelled agarose and acted on the cells. After one day of exposure to acetaminophen, the oil that separates the microdrops from one another is replaced with an aqueous phase as described in international application WO 2016/059302 to color the spheroids with fluorescent viability markers present in the aqueous phase. By taking an image of the final matrix, the spheroids whose viability was affected most could be determined. It was found that the higher the concentration of acetaminophen, the fewer cells survive. Thus, correlating the result for viability and the concentration of acetaminophen in the second microdrops, it could be determined that the range of toxicity of acetaminophen on these hepatic cells is between 10 and 30 mmol/L for a static exposure of 1 day.
The work that led to this invention received financing from the European Research Council in the context of the Seventh Framework Programme of the Union (FP7/2007-2013)/ERC grant agreement No. 278248.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1659418 | Sep 2016 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/074859 | 9/29/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/060471 | 4/5/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20100190263 | Srinivasan et al. | Jul 2010 | A1 |
| 20130078163 | Chung | Mar 2013 | A1 |
| 20150258543 | Baroud et al. | Sep 2015 | A1 |
| Number | Date | Country |
|---|---|---|
| 2 950 544 | Apr 2011 | FR |
| 2016059302 | Apr 2016 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20200038867 A1 | Feb 2020 | US |
