DEVICE AND METHOD FOR GENERATING AND COLLECTING DROPLETS

Abstract

 The present invention relates to a device (100) for generating droplets (41) comprising an emulsification junction (50) wherein the first input (51) is configured to be supplied with at least one first immiscible fluid (21-25), the second input (52) is configured to be connected to at least one first flow device (32) arranged to supply the second input (52) with a flow of a second immiscible fluid (31). The output (53) is connected to at least one second flow device (33). The device (100) is configured to operate a droplet generation mode wherein, the first input (51) is supplied by the second flow device (33), while the second input (52) is simultaneously supplied by the first flow device (32) so that droplet generation occurs and the generated droplets (41) are removed from the emulsification junction (50) by the second flow device (33).

Description

FIELD OF INVENTION

[0001] The present invention relates to the technical area of microfluidics. More particularly, the invention relates to a device for generating droplets comprising an emulsification junction. Such droplets may advantageously be used to generate pools of droplets, from a single or multiple reagents. For instance, an emulsion of identical droplets, or a high-diversity of droplet libraries, may be obtained.

BACKGROUND OF INVENTION

[0002] A "droplet library" is a set of microfluidic droplets, wherein the set includes at least two populations of identical droplets. Each population of identical droplets encapsulates at least one component with predetermined features. For instance, a first population of identical droplets may encapsulate a first drug with a first dosage and a second population of identical droplets may encapsulate a second drug with a second dosage.

[0003] A droplet library may advantageously be used in several applications such as high throughput screening. More particularly, the droplet library may be used to conduct thousands of parallel tests of different drugs and dosages to determine which drug and dosage is the most effective on a patient's disease.

[0004] The droplets may be generated by a process of emulsification which is a phenomenon that occurs when two immiscible fluids are mixed and droplets of one immiscible fluid are dispersed in the second immiscible fluid. The nanoliter to microliter-sized generated droplets are then collected into a vial, thus creating the droplet library.

[0005] A first solution to generate the droplets is to use a microarray spotter and to configure it to automatically deliver a predetermined quantity of at least one first fluid in a vial containing a second fluid that is immiscible with the first fluid. However, the microarray spotter lacks speed as it requires frequent washing and changes of tips, together with movements in the three dimensions of space. Moreover, it requires the droplets to travel through the air, which can be source of contamination, as well as lead to integration challenges.

[0006] Another solution is to use an emulsification junction, which is a system comprising two microfluidic input channels configured to carry two immiscible fluids toward an emulsification zone. The geometry of the intersection where the two immiscible fluids meet creates reproducible droplet breakup.

[0007] Several emulsification junctions of the type may be put in parallel to create different populations of droplets with different contents.

[0008] However, such systems often require frequent washings to avoid contamination, which is time-consuming. Moreover, such systems also require to use valves to collect or send the fluids in and out of the emulsification junction. This may cause distortion and/or contaminations of the droplets, notably through wetting in the valve internal volume, which might impair the droplets transport in a microfluidic system and the integrity of the related experiments.

[0009] Thus, the technical problem solved by the invention is to create an integrated device that is less prone to contamination between the droplets.

SUMMARY

[0010] To solve this problem, the Applicant has developed a device for generating droplets comprising an emulsification junction configured to generate droplets by putting into contact a flow of at least one first immiscible fluid with a flow of at least one second immiscible fluid. The emulsification junction comprises a first input, a second input, an output and an emulsification zone where the droplet generation occurs, wherein:

• the first input is configured to be supplied with the at least one first immiscible fluid,
• the second input is configured to be connected to at least one first flow device, said first flow device being arranged to supply the second input with the flow of the second immiscible fluid.

[0011] The invention is characterized in that the output is connected to at least one second flow device. The device is configured to operate a droplet generation mode wherein, the first input is supplied with the flow of said first immiscible fluid by the second flow device, while the second input is simultaneously supplied with the flow of said second immiscible fluid by the first flow device so that droplet generation occurs at the emulsification zone and the generated droplets are removed from the emulsification junction by the second flow device.

[0012] In other words, the second flow device is positioned downstream the emulsification junction output. In the droplet generation mode, the second flow device is configured to aspirate the flow of first immiscible fluid through the first input of the emulsification junction, while the first flow device pushes the second immiscible fluid through the second input of the emulsification junction. This way, droplets are generated at the emulsification zone and extracted through the output via the aspiration of the second flow device. Thus, during droplet generation mode, the droplets never have to travel through any valve. Indeed, inside a valve assembly, a droplet may wet on a surface present in the valve assembly. Once wetted on a surface, it may more easily merge with other droplets coming though the valve, thus forming droplets with different sizes. Controlling this wetting phenomenon is not possible inside a closed valve. Therefore, getting rid of valves allows to obtain uncontaminated droplets with identical shapes and sizes throughout the whole droplet generation process.

[0013] Advantageously, the device further includes:

• a closing device arranged upstream the first input, the closing device having an open state and a closed state, the closing device being configured to block the flow through the first input, and
• an outlet arranged downstream the output, the outlet having an open state and a closed state, the outlet being configured to allow extraction of the generated droplets from the device.

[0014] The device is further configured to operate a droplet collection mode wherein the closing device is in the closed state, while the outlet is in the open state, and a flow of the second immiscible fluid is generated to extract the generated droplets from the device through the opened outlet.

[0015] In other words, the device has two modes: a generation mode wherein the droplets are created in the emulsification junction and extracted out of the emulsification junction by an aspiration flow coming from downstream the emulsification junction, and a collection mode wherein the droplet generation is complete and the generated droplets are collected out of the device into a vial, for instance to create a droplet library. Therefore, the two modes cannot be activated at the same time.

[0016] To collect the droplets, a flow of the second immiscible fluid may be generated. According to an embodiment, the flow of the second immiscible fluid is generated by the second flow device. According to another embodiment, the flow of the second immiscible fluid is generated by the first flow device. The generated flow may either push or aspirate the generated droplets through the device outlet.

[0017] According to an embodiment, the emulsification junction includes a constriction comprising a constriction output, said constriction output being configured to lead the at least one first immiscible fluid to the emulsification zone.

[0018] According to another embodiment, the generated droplets may accumulate in a tube connected to the output of the emulsification junction. Alternatively, the device may further include an accumulator comprising an accumulation zone configured to trap the generated droplets, the accumulator being positioned downstream the output.

[0019] Advantageously, the accumulator is positioned between the output and the second flow device so that the second flow device may impulse a movement of the generated droplets toward the accumulation zone.

[0020] In practice, the accumulator is specifically designed to allow the generated droplets to accumulate. Advantageously, the accumulation zone is a dead volume wherein the droplets are led, either by a natural phenomenon such as buoyancy or gravity or by an external force such as a flow, a magnetic force, an acoustic force or a dielectrophoretic force.

[0021] According to a preferred embodiment, the accumulator is a gravity-based accumulator configured to trap the generated droplets that are driven by gravity in said accumulator.

[0022] The size of the accumulation zone is determined by the maximum number of droplets that is required to be generated.

[0023] Advantageously, the accumulator has a tapered shape comprising a base configured for the passage of the generated droplets and an apex where the accumulation zone is localized. In some embodiments, the apex includes the outlet. This way, the accumulated droplets are already close to the outlet and may rapidly be extracted from the device. Extraction may for instance be performed through a collection tubing, connected to the outlet and configured to lead the generated droplets into a vial. Alternatively, the generated droplets may be directly injected into another device, such as a microfluidic chip or a fluidic tubing.

[0024] According to another embodiment, the device further includes a third flow device connected to the outlet of the accumulator. The third flow device is configured to generate a flow configured to mix the generated droplets comprised inside the accumulation zone. In practice, the flow generated by the third flow device is configured to push the generated droplets towards the base of the accumulator, against the gravity, enabling the mixing of the droplets of different nature. This mixing occurs in a similar fashion than for a fluidized bed.

[0025] In a preferred embodiment, the second immiscible fluid is an oil phase chosen among fluorinated oil, silicon oil, vegetal oil, mineral oil, hydrocarbon oil and the first immiscible fluid is an aqueous phase comprising at least one among a cell culture medium, a drug solution, an antibody solution, a cell suspension, a suspension of organoids or cell tissues, an hydrogel solution, a microbead or particle suspension. The droplets may be stabilized with surfactants either in the oil phase (like PEG-di-Krytox fluorinated surfactant, perfluoro-octanol, perfluorodecanol, perfluoropolyether (PFPE) derivates including PFPE-PEG triblock copolymers, span 80, Abil EM90, monolein, oleic acid, n-butanol) or in the aqueous phase (phospholipid, Triton-X-100, SDS, Pluronic, Tween 20/80).

[0026] Alternatively, the second immiscible fluid may be an aqueous phase and the first immiscible fluid may be an oil phase.

[0027] In practice, the first input is configured to be supplied with a plurality of segments of the at least one first immiscible fluid, including at least a first segment and a second segment, two consecutive segments being separated with a third segment of the at least one second immiscible fluid.

[0028] According to the invention, a segment is a predetermined quantity of first immiscible fluid. Because the first immiscible fluids are miscible with each other, to keep then separated, it is possible to add a spacer plug made of a material that is immiscible with the first immiscible fluids. Such spacer plug may be made of the at least one second immiscible fluid used in the emulsification junction. Alternatively, it may be made of a third immiscible fluid to further avoid unwanted coalescence between different segments of the first immiscible fluid. These spacer plugs could be added in liquid (mineral oil, vegetable oil) or gaseous (bubbles of air or nitrogen, etc) states of matter.

[0029] Such plurality of segments may advantageously be obtained using a robotic arm configured to first draw a first segment of a first immiscible fluid, such as a chemotherapy or cell therapy fluid with a predetermined concentration, then draw a segment of the second immiscible fluid, such as oil, then draw a second segment of another first immiscible fluid, such as another type of chemotherapy or cell therapy with another concentration and so on. Alternatively, the second segment of first immiscible fluid may be made of the same first immiscible fluid as in the first segment of first immiscible fluid. The plurality of segments obtained may be transformed into droplets and collected into a vial, thus creating a droplet library that may be tested into a microfluidic device.

[0030] The device may be used to generate a suspension of identical droplets. Alternatively, the invention relates to the use of the device described above to generate a droplet library comprising at least two sets of droplets, wherein a first set of droplets is generated using a first type of first immiscible fluid and a second set of droplets is generated using a second type of first immiscible fluid. Such droplet library may advantageously be obtained using a single emulsification junction.

[0031] According to another aspect, the invention relates to a method for generating droplets using a device such as described above, wherein the method comprises the steps of:

• supplying the second input with a flow of at least one second immiscible fluid, the second input being configured to carry the second immiscible fluid toward the emulsification zone, and
• simultaneously supplying the first input with a flow of at least one first immiscible fluid by aspirating said first immiscible fluid from downstream the output, the first input being configured to carry the first immiscible fluid toward the emulsification zone, so that droplets are generated at the emulsification zone, the generated droplets being aspirated out of the emulsification junction by said aspiration from downstream the output.

[0032] Advantageously, the method further comprises the steps of accumulating the generated droplets in an accumulator such as described above.

[0033] According to another embodiment, the invention also relates to a method for collecting droplets from the device described above, wherein the method comprises the steps of:

• closing the first input using the closing device,
• opening the outlet, and
• supplying a flow of a second immiscible fluid so that the generated droplets are extracted out of the device through the opened outlet.

[0034] Advantageously, the flow of a second immiscible fluid is supplied from downstream the output.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 

Figure 1 is a schematic front view representation of the device according to an embodiment of the invention in the droplet generation mode,

Figure 2 is a is a schematic front view representation of the device according to an embodiment of the invention in the droplet collection mode,

Figure 3 is a schematic cross section representation of an emulsification junction according to an embodiment of the invention,

Figure 4 is a schematic cross section representation of the emulsification junction from figure 3, zoomed on the constriction,

Figure 5 is a schematic top view of an emulsification junction according to an embodiment of the invention,

Figure 6 is a diagram representative of the steps of the method for generating droplets using a device according to an embodiment of the invention,

Figure 7 is a diagram representative of the steps of the method for collecting droplets using a device according to an embodiment of the invention,

Figure 8 is a schematic front view representation of the first step of the method from figure 5, in which a first segment of first immiscible fluid is aspirated in the device according to an embodiment of the invention,

Figure 9 is a schematic front view representation of the first step of the method from figure 5, in which a first segment of second immiscible fluid is aspirated in the device according to an embodiment of the invention,

Figure 10 is a schematic front view representation of the first step of the method from figure 5, in which several segments of first and second immiscible fluid have been aspirated in the device according to an embodiment of the invention,

Figure 11 is a schematic front view representation of the first step of the method from figure 5, in which the segments are driven toward the emulsification junction,

Figure 12 is a schematic front view representation of the second and third steps of the method from figure 5,

Figure 13 is a schematic front view representation of the fourth step of the method from figure 5,

Figure 14 is a schematic front view representation of the first step of the method from figure 5,

Figure 15 is a schematic front view representation of the of the first step of the method from figure 5,

Figure 16 is a schematic front view representation of the steps from the method in figure 6, and

Figure 17 is a graph with experimental pictures showing the effect of the constriction of the droplet emulsification junction on the droplet volumes generated from a segment.

DETAILED DESCRIPTION

[0036] As illustrated on figure 1 and 2, the device 100 according to the invention includes an emulsification junction 50 with a first input 51, a second input 52 and an output 53.

Emulsification junction

[0037] An emulsification junction 50 is a system comprising two inputs 51, 52 configured to carry at least one first immiscible fluid 21-25 and at least one second immiscible fluid 31 toward an emulsification zone 55. The geometry of the emulsification zone 55 where the at least two immiscible fluids 21-25, 31 meet and droplets breakup occurs.

[0038] The emulsification junction 50 may be of any type such as a T-junction, a Y-junction, a flow-focusing junction, a co-flow focusing junction, a slope emulsification junction, a step emulsification junction, a buoyancy-emulsification junction, a centrifugal step emulsification junction, an electrohydrodynamic emulsification junction, or an acoustic emulsification. In particular, as illustrated in figures 3 and 4, the emulsification junction 50 may advantageously be a 3D flow focusing junction, where the first and second immiscible phases are forced through narrow openings of the collection duct simultaneously, resulting in the uniform break-up of the first phase while surrounded by the second phase. Such emulsification junction 50 may optionally include a constriction 54.

[0039] A 3D flow focusing junction is made of at least two ducts 56 57, wherein at least a first duct 56 is positioned inside at least a second duct 57. Advantageously, the ducts 56, 57 are concentrical. The ducts 56, 57 may be capillaries that may be made of glass, plastic, resin or fluorinated polymers. The 3D flow focusing junction comprises a first duct 56 including a first input 51, a second input 52 and an output 53. The first input 51 and the output 53 are preferably aligned along a right d2, while the second input 52 is angled with respect to right d2 and aligned along right d3.

[0040] The first input 51 and the second input 52 may have an inner diameter comprised between 0.1 mm and 2 mm, preferably 1 mm and an external diameter preferably comprised between 0.2 mm and 3 mm, preferably 1.6 mm. The output 53 has a smaller inner diameter that may be comprised between 0.1 mm and 2 mm, preferably 0.75 mm and an external diameter preferably comprised between 0.2 mm and 3 mm, preferably 1.6 mm.

[0041] The second duct 57 is positioned inside the first duct 56, along right d2. It comprises a first end 59 and a second end 58. It preferably has an inner diameter comprised between 0.1 mm and 1 mm, preferably 0.25 mm and an external diameter preferably comprised between 0.5 mm and 1.6 mm, preferably 0.75 mm. The length of the second duct 57 is advantageously comprised between 25 mm and 75 mm.

[0042] Preferably, the inner diameter of the output 53 is equal to the external diameter of the second duct 57. This way, a fluid-proof cavity is created between the inner diameter of the first duct 56 and the external diameter of the second duct 57, wherein a fluid may flow.

[0043] An emulsification zone 55 configured for the droplet generation may be defined at the front of the second duct first end 59.

[0044] Advantageously, the emulsification junction 50 of the invention includes a constriction 54, comprising a first end 541 and a second end 542, positioned in front of the second duct first end 59. In that case, the emulsification zone 55 may be defined between the constriction 54 and the second duct first end 59.

[0045] Geometry and location of the constriction 54, with respect to the emulsification zone 55, play a role in obtaining a desired droplet size and droplet uniformity.

[0046] For instance, as illustrated in figure 4, a cylindrical shaped constriction may be used. In figure 4, d1 defines the inner diameter of the constriction second end 542, L defines the length of the constriction 54, and w defines the distance between the constriction second end 542 and the second duct first end 59.

[0047] The constriction 54 geometry is constrained by the desired droplet size. The constriction inner diameter d1 is preferably equal to, or smaller than, the desired droplet diameter D. In other words, it is possible to define the following formula: d1 ≲ D. The constriction length L is preferably equal to, or greater than, the desired droplet diameter D. In other words, it is possible to define the following formula: L ≳ D. The gap between the constriction end and the emulsification capillary end is preferably less than ten times the desired droplet diameter D. In other words, it is possible to define the following formula: w ≲ 10D.The inner diameter of the constriction 54 may be constant along the length of the constriction or alternatively, it may have a tapered shape with a bigger diameter at the constriction first end 541 and a smaller diameter at the constriction second end 542.

[0048] The constriction 54 allows to minimize the polydispersity among the segments of first immiscible fluid 23 in the first input 51. The presence of a constriction 54 decreases the difference between the cross-section diameter of the first input 51, which is typically of 1mm, and the cross-section diameter of the second duct first end 59, which is typically of 250 µm. The constriction 54 prevents the formation of a large droplet 47 in the last set of droplets, at the end of the first input 51. This improvement can be seen in figure 16, where a constriction with an inner diameter d1 of 500µm was used.

[0049] Figure 5 shows an example of an emulsification junction 50 that may be obtained by 3D printing. More precisely, the emulsification junction 50 may take the shape of a microfluidic ship comprising a body 73 made of a rigid material configured to maintain together the ducts 56, 57. The ducts 56, 57 may also be printed using a plastic such as PolyEtherEtherKetone (PEEK) or tetrafluoroethylene (TEFLON) or a combination thereof. Silicon tubes 71 and glue 72 may be used to maintain/assemble the ducts 56, 57 together. The use of silicon tubes 71 and glue 72 enables better concentric alignment of the ducts 56, 57, and a stable control over the distance w in order to allows reproducibility in droplet production.

[0050] The body 73 may comprise a lumen 74 configured to allow visual access to the emulsification zone 55 and to allow direct microscopy measurements to be performed, for instance of the distance w. The lumen 74 may be an opening or filled with a transparent material such as glass, plastic, or Polydimethylsiloxane (PDMS).

First input 51

[0051] As illustrated in figure 1, for the purpose of the droplet generation mode, the first input 51 is configured to be supplied with at least one first immiscible fluid 21-25. To that end, the first input 51 may be connected to at least one first tank 61 containing the at least one first immiscible fluid 21-25. In an embodiment, the first input 51 may be in permanence connected to several first tanks 61 containing different first immiscible fluids 21-25. A device may therefore be used to select the desired first tank 61, such as a multi-way directional control valve.

[0052] Alternatively, a device may be used to operate the connection between the desired first tank 61 and the first input 51 when needed, such as a robotic arm movable in at least two directions of space.

[0053] In a first embodiment, the first input 51 may be supplied with a continuous flow of first immiscible fluid 21-25. Alternatively, the first input 51 may be supplied with segments of different first immiscible fluids 21-25, separated by segments of second immiscible fluid 31, supplied by a second tank 62.

[0054] The connection between the first input 51 and the at least one first and second tanks 61, 62 may be obtained using tubular connections 12 such as flexibles tubes made of caoutchouc, Polyvinyl chloride (PVC) and the like.

[0055] The aspiration of the at least one first immiscible fluid 21-25 inside the tubular connections may be performed either by the at least one first flow device 32 or the at least one second flow device 33.

[0056] For the purpose of the droplet collection mode, as illustrated in figure 2, a closing device 26 may be arranged upstream the first input 51. The closing device 26 may be a valve, a plug or any device configured to block the flow through the first input 51. The closing device 26 may be made of elastic materials, such as rubber or PDMS to facilitate rapid opening and/or closing. The closing device 26 may be operated manually or electromechanically, and may be able to withstand at least 10 mbar of internal pressure when in closed position.

Second input 52

[0057] As illustrated on figure 1 and 2, the second input 52 is configured to be connected to at least one first flow device 32, said first flow device 32 being arranged to supply the second input 52 with a flow of at least one second immiscible fluid 31. The first flow device 32 may also be configured to aspirate a flow coming from the emulsification junction 50.

[0058] The connection between the second input 52 and the at least one first flow device 32 may be obtained using tubular connections 13 such as flexibles tubes made of caoutchouc, Polyvinyl chloride (PVC) and the like.

[0059] The first flow device 32 may be a 0.1 mL to 50 mL syringe pump, a peristaltic pump, a pressure-driven flow generator using flow sensor, a pressure-driven flow generator using flow resistance, a hydrostatic pressure driven flow generator and the like.

[0060] In a first embodiment, the first flow device 32 comprises a tank configured to contain a first type of second immiscible fluid 31. Alternatively, the first flow device 32 may comprise several tanks containing different types of second immiscible fluids 31. The first flow device 32 may be configured to alternatively provide a specific type of second immiscible fluid 31 by selecting the correct tank. Alternatively, the second input 52 may be connected to several first flow devices 32 containing each a tank filled with a specific type of second immiscible fluid 31. Depending on the required second immiscible fluid 31, the correct first flow device 32 may be selected to supply the second input 52 in second immiscible fluid 31. Moreover, the at least one first flow device 32 may comprise a tank configured to receive the fluid aspirated from the emulsification junction 50.

Output 53

[0061] As illustrated on figure 1 and 2, the output 53 is configured to be connected to at least one second flow device 33, said first flow device 33 being arranged to either supply a flow of at least one second immiscible fluid 31 or to aspirate a flow coming from the emulsification junction 50.

[0062] The connection between the output 53 and the at least one second flow device 33 may be obtained using tubular connections 14 such as flexibles tubes made of rubber, silicone, Polyvinyl chloride (PVC) or any other elastic material and the like.

[0063] The second flow device 33 may be a 0.1 mL to 50 mL syringe pump, a peristaltic pump, a pressure-driven flow generator using flow sensor, a pressure-driven flow generator using flow resistance, a hydrostatic pressure driven flow generator...

[0064] In a first embodiment, the second flow device 33 comprises a tank configured to contain a first type of second immiscible fluid 31. Alternatively, the second flow device 33 may comprise several tanks containing different types of second immiscible fluids 31. The second flow device 33 may be configured to alternatively provide a specific type of second immiscible fluid 31 by selecting the correct tank. Alternatively, the second input 52 may be connected to several second flow devices 33 containing each a tank filled with a specific type of second immiscible fluid 31. Depending on the required second immiscible fluid 31, the correct second flow device 33 may be selected to supply the second immiscible fluid 31. Moreover, the at least one second flow device 33 may comprise a tank configured to receive the fluid aspirated from the emulsification junction 50.

Accumulator 15

[0065] As illustrated on figure 1 and 2, the device 100 according to the invention may advantageously include an accumulator 15 comprising an accumulation zone 17 configured to trap the generated droplets 41-45. The accumulator 15 is positioned downstream the output 53, preferably between the output 53 and the second flow device 33.

[0066] The accumulator 15 may have a tapered shape comprising a base 151 and an apex 154. The base 151 advantageously comprises at least one opening 152 configured for the passage of the generated droplets 41-45 coming from the output 53. The accumulator 15 may also comprise a second opening 153 configured for the passage of a flow of the second immiscible fluid 31.

[0067] For the purpose of the droplet collection mode, as illustrated in figure 2, an outlet 16 may be arranged downstream the output 53.

[0068] The outlet 16 is configured for the extraction of the generated droplets 41-45 out of the device 100, for instance into a vial 101. Preferably, the outlet 16 is included at the apex 154 of the accumulator 15. The outlet 16 may be a sealable opening with an open state and a closed state. The outlet 16 may for instance be sealed using a valve, a plug or the like. The outlet 16 may also include a tubular connection 161 configured to carry the generated droplets 41-45 directly into the vial. The outlet 16 may be closed using a valve made of elastic materials such as rubber, PDMS, to facilitate rapid opening/closing. It may be operated manually or electromechanically, and may be able to withstand at least 10 mbar of internal pressure when in closed position.
Alternatively, the accumulator may have other shapes such as an asymmetrical conical shape, a spherical shape, cylindrical shape, an ellipsoidal shape, and a combination thereof. Alternatively, the openings 151 and 152 may be positioned on a lateral wall of the accumulator 15. Other accumulator geometry examples may include all geometries with a smooth transition from a large cross-section are to a small orifice, such that it leaves no space inside the accumulator where at least one droplet may remain trapped after the droplet extraction.

[0069] The typical dimension ranges for an accumulator 15 with a tapered shape are the following. The base 151 preferably has a diameter comprised between 1mm and 50mm. The height of the accumulator 15 is preferably comprised between 10mm and 10cm.

Method

[0070] As illustrated on figures 5 and 6, according to another aspect, the invention relates to a method 200for generating droplets using a device 100 such as described above and to a method for collecting said droplets 300.

[0071] The first step of the method for generating droplets 200 is to supply 202 the first input 51 with an input flow of at least one first immiscible fluid 21-24.

[0072] According to a preferred embodiment, illustrated in figures 7 to 15, the flow of at least one second immiscible fluid 31 comprises a plurality of segments of different first immiscible fluids 21-24, separated by segments of the at least one second immiscible fluid 31.

[0073] For instance, the plurality of segments may be prepared using a robotic arm movable in the three directions of space. As illustrated in figure 7, the robotic arm may move a tubular connection 12 into a first well 64 comprised in a well-plate 63. The well 64 contains a first type of first immiscible fluid 21. A first segment of the first immiscible fluid 21 may be aspirated. In this example, the aspiration is engendered by the first flow device 33.

[0074] Then, as illustrated in figure 8, the robotic arm may move the tubular connection 12 into a tank 62 containing a second immiscible fluid 31. A first segment of the second immiscible fluid 31 may be aspirated.

[0075] In a third movement, the robotic arm may move the tubular connection 12 into a second well comprised in the well-plate 63 and containing a second type of first immiscible fluid 22. In a fourth movement, the robotic arm may move the tubular connection 12 again into tank 62 to aspirate a second segment of second immiscible fluid 31.

[0076] These movements may be repeated indefinitely to make multiple segments of first immiscible fluid 21-24. For instance, a number comprised between 2 and 20 segments of first immiscible fluid 21-24 may be present in the tubular connection 12 such as illustrated in figure 9. In an embodiment, the samples are directly collected from a well-plate 63, such as illustrated in figure 7.

[0077] In a last movement, the tubular connection 12 is left, by the robotic arm, into tank 62 to allow aspiration of the second immiscible fluid 31. Therefore, while the second immiscible fluid 31 is being aspirated, the segments of first immiscible fluid 21 24 begin to move up the tubular connection 12 toward the emulsification junction 50, as illustrated in figure 10.

[0078] The typical size of a segment of first immiscible fluid 21-24 is comprised between 1 µL and 1 mL. The size of a segment of first immiscible fluid 21-24 may vary from one another depending on the desired number of generated droplets. The typical size of a segment of second immiscible fluid 31 is comprised between 1 µL and 1 mL.

[0079] The at least one second immiscible fluid 31 may be an organic solvent, such as Hydrofluoroethers combined with surfactants. Alternatively, the at least one second immiscible fluid 31 may be an oil phase chosen among fluorinated oil, silicon oil, vegetal oil, mineral oil, hydrocarbon oil The droplets may be stabilized with surfactants either in the oil phase (like PEG-di-Krytox fluorinated surfactant, perfluoro-octanol, perfluorodecanol, perfluoropolyether (PFPE) derivates including PFPE-PEG triblock copolymers, span 80, Abil EM90, monolein, oleic acid, n-butanol) or in the aqueous phase (phospholipid, Triton-X-100, SDS, Pluronic, Tween 20/80).The first immiscible fluid 21-24 is preferably an aqueous phase comprising at least one among a cell culture medium, a drug solution, an antibody solution, a cell suspension, a suspension of organoids or cell tissues, an hydrogel solution, a microbead or particle suspension.

[0080] As illustrated in figure 11, once the first segment of first immiscible fluid 21 has entered the emulsification junction first opening 51, the second input 52 is simultaneously supplied, in a second step 202, with a flow of the at least one second immiscible fluid 31 using the first flow device 32, while aspiration with the second flow device 33 continues.

[0081] As illustrated in figure 11 and 12, droplets 41 of the first type of immiscible fluid 21 are then generated, in a third step 203, at the output 53 of the emulsification junction 50. Once the first segment of first immiscible fluid 21 completely transformed into droplets 41, the segment of second immiscible fluid 31 passes through the emulsification junction 50 and washes out the remaining droplets 41. Then, a second segment of first immiscible fluid 22 enters the emulsification junction first opening 51 and is transformed into droplets 42 of the second type of immiscible fluid 22. This phenomenon continues up until there is no segment of first immiscible fluid 21-24 left inside the tubular connection 12.

[0082] The generated droplets 41-45 may have a size comprised between 2 nL and 2 µL depending on the geometry of the ducts used in the emulsification junction 50.

[0083] The typical flow rates used to supply the emulsification junction 50 in first immiscible fluid 21-25 and second immiscible fluid 31 may be comprised between 10-1000 µL/min. The number of droplets 41-45 generated may be comprises between 5 and 100 000.

[0084] While the segments of first immiscible fluid 21-24 are transformed into droplets 41-44, said generated droplets 41-45 tend to migrate toward the accumulator 15 and accumulate, in a fourth step 204, into the accumulation zone 17, due to buoyancy.

[0085] Once all the generated droplets 41-44 trapped into the accumulation zone 17, the first flow device 32 and second flow device 33 are stopped as illustrated in figure 14. The droplet generation mode is complete.

[0086] Advantageously, the device 100 may include a third flow device (not shown in the figures) connected to the outlet 16 of the accumulator 15. The third flow device may be configured to generate a flow configured to mix the generated droplets comprised inside the accumulation zone 17. In practice, the flow generated by the third flow device is configured to push the generated droplets towards the base of the accumulator 15, against gravity, enabling the mixing of the droplets of different nature. This mixing occurs in a similar fashion than for a fluidized bed.

[0087] For the droplet collection mode, the tubular connection 12 end is plugged in a first step 301, using the closing device 26, while the outlet 16 is opened in a second step 302. The second flow device 33 is activated to supply, in a third step 303, a flow of second immiscible fluid 31 thus pushing the generated droplets 41-44 through the outlet 16 and into a vial 101. A droplet library is then created. The size of the library created may be of 0.1 mL to 10 mL.

[0088] The device and method of the invention may be used for drug screening. To this end, a library of aqueous droplets may be prepared with different compounds such as drugs, including chemotherapies, cells, and biologics to test on cells. Such cells may be cancer cells collected from a patient.

[0089] The device and method of the invention may also be used to test culture conditions. To this end, a library of different medium composition may be prepared by varying the viscosity, the type and concentration of serums, the growth factors, the amino acids, the buffers, the addition of small quantities of hydrogel molecules, or molecules that favor the 3D aggregation of cells.

[0090] The device and method of the invention may also be used for cell encapsulation. To this end, cells may be encapsulated from a single or several sources. This could be used to screen multiple patient samples.

Claims

1. Device (100) for generating droplets (41-45) comprising an emulsification junction (50) configured to generate droplets (41-45) by putting into contact a flow of at least one first immiscible fluid (21-25) with a flow of at least one second immiscible fluid (31), the emulsification junction (50) comprising a first input (51), a second input (52), an output (53) and an emulsification zone (55) where the droplet generation occurs, wherein:

- the first input (51) is configured to be supplied with the at least one first immiscible fluid (21-25),

- the second input (52) is configured to be connected to at least one first flow device (32), said first flow device (32) being arranged to supply the second input (52) with the flow of the second immiscible fluid (31),

characterized in that the output (53) is connected to at least one second flow device (33), the device (100) being configured to operate a droplet generation mode wherein, the first input (51) is supplied with the flow of said first immiscible fluid (21-25) by the second flow device (33), while the second input (52) is simultaneously supplied with the flow of said second immiscible fluid (31) by the first flow device (32) so that droplet generation occurs at the emulsification zone (55) and the generated droplets (41-45) are removed from the emulsification junction (50) by the second flow device (33).

2. Device according to claim 1, wherein the device (100) further includes:

- a closing device (26) arranged upstream the first input (51), the closing device (26) having an open state and a closed state, the closing device (26) being configured to block the flow through the first input (51),

- an outlet (16) arranged downstream the output (53), the outlet (16) having an open state and a closed state, the outlet (16) being configured to allow extraction of the generated droplets (41-45) from the device (100),

the device (100) being further configured to operate a droplet collection mode wherein the closing device (26) is in the closed state, while the outlet (16) is in the open state, and a flow of the second immiscible fluid (31) is generated to extract the generated droplets (41-45) from the device (100) through the opened outlet (16).

3. Device according to any of claim 1 to 2, wherein the first input (51) is configured to be supplied with a plurality of segments of the at least one first immiscible fluid (21-25), including at least a first segment and a second segment, two consecutive segments being separated with a third segment of the at least one second immiscible fluid (31).

4. Device according to any of claim 1 to 3, wherein the device (100) further includes an accumulator (15) comprising an accumulation zone (17) configured to trap the generated droplets (41-45), the accumulator (15) being positioned downstream the output (53).

5. Device according to claim 4, wherein the accumulator (15) is positioned between the output (53) and the second flow device (33).

6. Device according to either claim 4 or 5, wherein the accumulator (15) is a gravity-based accumulator (15) configured to trap the generated droplets (41-45) that are driven by gravity in said accumulator (15).

7. Device according to any of claim 4 to 6, wherein the accumulator (15) has a tapered shape comprising a base configured for the passage of the generated droplets (41-45) and an apex where the accumulation zone (17) is localized.

8. Device according to claims 2 and 7, wherein the apex includes the outlet (16).

9. Device according to any of claim 1 to 8, wherein the emulsification junction (50) includes a constriction (54) comprising a constriction output, said constriction output being configured to lead the at least one first immiscible fluid (21-25) to the emulsification zone (55).

10. Use of the device according to any of claim 1 to 9 to generate a droplet library comprising at least two sets of droplets (41-45), wherein a first set of droplets (41-45) is generated using a first type of first immiscible fluid (21-25) and a second set of droplets (41-45) is generated using a second type of first immiscible fluid (21-25).

11. Method (200) for generating droplets using a device (100) according to any one of claims 1 to 9, wherein the method comprises the steps of:

- supplying (201) the second input (52) with a flow of at least one second immiscible fluid (31), the second input (52) being configured to carry the second immiscible fluid (31) toward the emulsification zone (55), and

- simultaneously supplying (202) the first input (51) with a flow of at least one first immiscible fluid (21-25) by aspirating said first immiscible fluid (21-25) from downstream the output (53), the first input (51) being configured to carry the first immiscible fluid (21-25) toward the emulsification zone (55), so that droplets (41-45) are generated at the emulsification zone (55), the generated droplets (41-45) being aspirated out of the emulsification junction (50) by said aspiration from downstream the output (53).

12. Method according to claim 11, wherein the method further comprises the steps of accumulating the generated droplets (41-45) in an accumulator (15) according to claims 5 to 9.

13. Method for collecting droplets from the device (100) according to any one of claims 1 to 9, wherein the method comprises the steps of:

- closing (301) the first input (51) using the closing device (26),

- opening (302) the outlet (16), and

- supplying (303) a flow of a second immiscible fluid (31) so that the generated droplets (41-45) are extracted out of the device (100) through the opened outlet (16).

14. Method for collecting droplets from the device (100) according to claim 13, wherein the flow of a second immiscible fluid (31) is supplied from downstream the output (53).

Drawing