Method of forming a multilayer aggregate of objects

Abstract

A method of forming a multilayer aggregate of objects in a channel including a liquid, the method including: a) providing objects at first and second superposed regions of the channel, b) obtaining first and second aggregates of objects, optionally by applying transverse acoustic waves, preferably stationary waves, within each region to objects, and c) bringing the first and second aggregates into contact to form the multilayer aggregate of objects by submitting the first and second aggregates to: gravity in absence of acoustic waves, or to acoustic waves, optionally stationary waves, inducing displacement of the first and second aggregates toward each other.

Claims

1. A method of forming a multilayer aggregate of objects in a channel comprising a liquid, said method comprising: a) providing objects at first and second superposed regions of the channel, b) obtaining first and second aggregates of objects within each region, and c) bringing said first and second aggregates into contact to form said multilayer aggregate of objects by submitting said first and second aggregates to: gravity in absence of acoustic waves, or to acoustic waves inducing displacement of said first and second aggregates toward each other.

2. The method according to claim 1, wherein: in step b), the first aggregate being obtained by applying transverse acoustic waves to the objects within the first region, the second aggregate being obtained by applying transverse acoustic waves to the objects within the second region and the acoustic waves used at step b) being generated along a thickness of the channel, or in step c) said first and second aggregates being brought into contact to form said multilayer aggregate of objects by submitting said first and second aggregates to acoustic waves and the acoustic waves used at step c) being generated along a thickness of the channel.

3. The method according to claim 1, in step b), the first aggregate being obtained by applying transverse acoustic waves to the objects within the first region, the second aggregate being obtained by applying transverse acoustic waves to the objects within the second region and in step c) said first and second aggregates being brought into contact to form said multilayer aggregate of objects by submitting said first and second aggregates to acoustic waves, step c) comprising submitting the aggregates to acoustic waves having at least one of: a different number of acoustic nodes than the acoustic waves applied at step b), at least one acoustic node having a transverse position which is different from the transverse position(s) of the acoustic node(s) of the acoustic waves applied at step b), at least one acoustic antinode having a transverse position which is different from the transverse position(s) of the acoustic antinode(s) of the acoustic waves applied at step b), and an amplitude less than the amplitude of the acoustic waves applied at step b).

4. The method according to claim 1, the channel comprising, over at least a portion of its length, a wall whose thickness varies.

5. The method according to the claim 4, in step c) said first and second aggregates being brought into contact to form said multilayer aggregate of objects by submitting said first and second aggregates to acoustic waves, at least a first and a second acoustic field generator being present along the length of the channel and: said first and second acoustic field generator emitting acoustic waves at step b) for obtaining aggregates of first and second objects and emitting the acoustic waves at step c), and said channel having first and second walls respectively situated opposite to said first and second acoustic field generators which have different thicknesses in such a manner that the acoustic waves applied at step c) have a node or an antinode which is located on said second wall.

6. The method according to claim 1, the width or thickness of the channel varying over at least a portion of its length.

7. The method according to claim 6, in step c) said first and second aggregates being brought into contact to form said multilayer aggregate of objects by submitting said first and second aggregates to acoustic waves and, in step b), the first aggregate being obtained by applying transverse acoustic waves to the objects within the first region and the second aggregate being obtained by applying transverse acoustic waves to the objects within the second region, at least a first and a second acoustic field generator being present along the length of the channel for generating respectively the acoustic waves at steps b) and c), wherein: said first and second acoustic field generators emit acoustic waves at steps b) and c) which have substantially a same main frequency, and the width or thickness of the channel varies over at least a portion of its length in such a manner that the acoustic waves applied at step c) have a different number of acoustic nodes than the acoustic waves applied at step b).

8. The method according to claim 1, in step c) said first and second aggregates being brought into contact to form said multilayer aggregate of objects by submitting said first and second aggregates to acoustic waves and, in step b), the first aggregate being obtained by applying transverse acoustic waves to the objects within the first region and the second aggregate being obtained by applying transverse acoustic waves to the objects within the second region, a main frequency of the acoustic waves applied at step c) being different from a main frequency of the acoustic waves applied at step b) in such a manner that the acoustic waves applied at step c) have a different number of acoustic nodes than the acoustic waves applied at step b).

9. The method according to claim 1 comprising: a) providing objects at first, second and third superposed regions of the channel, b) obtaining first, second and third aggregates of objects within each region to objects, and c) putting said first, second and third aggregates in contact in order to form said multilayer aggregate of objects by submitting said first, second and third aggregates to: gravity in absence of acoustic waves, or to acoustic waves inducing forces displacing said first, second and third aggregates toward each other.

10. The method according to claim 1, wherein layers of objects at the first and second superposed regions of the channel are provided at step a).

11. The method according to a claim 1, wherein the objects present in the first region, at step a), have a different size or nature from objects present, at step a), in the second region.

12. The method according to claim 1, objects being mono or polydisperse biological cells, stem cells or primary cell lines.

13. The method according to claim 1, said objects having a positive acoustic contrast factor.

14. The method according to claim 1, said objects having a negative acoustic contrast factor.

15. The method according to claim 14, said objects being selected from the group consisting of bubbles, microbubbles, nanobubbles, droplets, fat cells, blood lipids, phospholipid vesicles, and mixtures thereof.

16. The method according to claim 1, the method comprising after step b) a determination of at least one feature of the objects.

17. The method according to claim 1, the method comprising after step b) a step of coalescence of the objects.

18. The method according to claim 1, said objects being evacuated outside the channel after step c).

19. The method according to claim 1, at least one of the first and second aggregates comprising at least 100 objects.

20. The method according to claim 1, wherein: in step b), the first aggregate being obtained by applying transverse acoustic waves to the objects within the first region and the second aggregate being obtained by applying transverse acoustic waves to the objects within the second region, a plurality of acoustic wave generators being present along the length of the channel for generating said acoustic waves at step b), or in step c), said first and second aggregates being brought into contact to form said multilayer aggregate of objects by submitting said first and second aggregates to acoustic waves and a plurality of acoustic wave generators being present along the length of the channel for generating said acoustic waves at step c).

21. The method according to claim 1, the liquid being flowing at least during steps b) or c).

22. The method according to claim 1, the liquid not being flowing during step b).

23. The method according to claim 1, all or part of the method being carried out in a gravitational field of strength lower than or equal to 10 m/s.sup.2.

24. The method according to claim 1, all or part of the method being carried out in a gravitational field of strength substantially different from the strength of the gravitational field at the Earth's surface.

25. The method according to claim 1, wherein: the liquid is a cell culture medium, and at least one of the first and second aggregates comprises cells, and said cells being cultured while present in the at least one of said first and second aggregates.

26. The method according to claim 25, at least one tissue being formed by the cell culture.

27. The method according to claim 26, a bone tissue being formed by the cell culture.

28. The method according to claim 27, some cell culture medium being introduced at least once in the channel during the cell culture.

29. The method according to claim 27, the cell culture medium being renewed at least once during the cell culture.

30. The method according to claim 1, the first and second aggregates being kept during at least one hour.

31. The method according to claim 1, the first and second aggregates being 3D aggregates.

32. The method according to claim 1, a plurality of sets of first and second aggregates being present along a longitudinal axis of the channel.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be better understood from a reading of the detailed description below, of non-limitating examples for its implementation, and from examination of the attached drawings, in which:

(2) FIG. 1 shows a device for carrying out of a method according to the invention,

(3) FIG. 2 shows an aggregate obtained according to a method of the invention,

(4) FIG. 3 is a view according to III-III of the channel used in FIG. 1,

(5) FIG. 4 is an example of evacuation of the multilayer aggregate formed according to the method shown in FIG. 1,

(6) FIGS. 5 to 8 are variants of devices for performing methods according to the invention,

(7) FIG. 9 is an embodiment of a channel that may be used in the methods according to the invention, and

(8) FIGS. 10 to 12 B show experimental results obtained by the methods according to the invention.

DETAILED DESCRIPTION

(9) FIG. 1 shows a device 1 which may be used in the methods according to the invention. The device 1 comprises a channel 2 extending along a longitudinal axis X.

(10) The channel 2 may, as mentioned above, be a microchannel.

(11) The channel 2 presents a cross-section that is for example rectangular. In the example described, the length/thickness ratio of the channel 2 is greater than 10.

(12) The channel 2 has bottom and top walls 3 and 4. The channel 2 is as shown in fluidic communication with inlets 7, for example five inlets 7.

(13) The inlets 7 open out in the top and bottom walls 4 and 3 of the channel 2 and four inlets 7 open out perpendicularly to the X axis as shown. Further, the inlet 7 that opens out in the top wall 4 of the channel 2 opens out into said channel 2 parallel to the X axis.

(14) In a variant not shown, the inlets all open out into the bottom wall of the channel.

(15) The inlets 7 are of substantially rectangular section, and of width equal to the width of the channel 2.

(16) Inlets 7 are separated by transverse separation walls 10 that are perpendicular to the longitudinal axis X.

(17) The inlets 7 present as shown thicknesses that are smaller than that of the channel 2, e.g. less than half its thickness.

(18) In the embodiment shown in FIG. 1, some of the inlets 7 may be offset along the X axis such as separation walls 10.

(19) At least one inlet 7 opens out, as shown, into a first zone 45 connected to a second zone 46 of greater thickness into which another inlet 7 opens out, the thicknesses being measured along the thickness of the channel 2.

(20) The second zone 46 is connected to a central zone 47 of the channel 2.

(21) The differences in thickness between the zones 45 and 46, and between the zones 46 and 47, correspond to the difference in height between the separation walls 10.

(22) The device 1 can be used as follows in the methods according to the invention.

(23) As shown in FIG. 1 for example, a carrier liquid L and a plurality of objects O are caused to flow via the inlets 7.

(24) Objects O may be mono or polydisperse, said objects O may be biological cells and liquid L may be a biological liquid such as, e.g. blood.

(25) Where appropriate, the device 1 may have one or more valves for controlling the injection of objects O via the inlets 7, where such a valve may, for example, be a solenoid valve presenting a single passage or multiple passages.

(26) The injection of objects O via the inlets 7 can be controlled in frequency and in flow rate so as to enable the device 1 to operate continuously in order to process large volumes of objects.

(27) Such a device 1 provides objects O at first 50 and second 60 superposed regions of the channel 2 (step a)) by a hydrodynamic focusing technique.

(28) First 51 and second 61 layers of objects O are respectively present in said first 50 and second 60 superposed regions, the thickness of said first 51 and second 61 layers being controlled by hydrodynamic focusing.

(29) The first 51 and second 61 layers of objects O are, as shown in FIG. 1, separated by a layer of pure liquid L. The objects O present in the first layer 51 may be identical to the objects O present in the second layer 61. In a variant, the objects O present in the first layer 51 and the objects O present in the second layer 61 belong to different species. In a variant, the objects O present in the first layer 51 have a different size from objects O present in the second layer 61.

(30) Such layers of objects may undergo hydrodynamic shear induced diffusion [3] that can be detrimental for multi-levitation under-flow.

(31) Thus, the device 1 is provided with acoustic field generators 100a and 100b which are, as shown, fastened to the top wall 4 of the channel 2. In FIG. 1, the acoustic field generators 100a and 100b are arranged along the channel 2 and present on the same side of said channel 2. In a variant not shown, the acoustic field generators may be arranged along the channel and present on opposite sides of said channel.

(32) The acoustic field generator 100a enables formation, at step b), of first 110 and second 111 aggregates of objects O. As shown in FIG. 1, the objects O are placed in multi-levitation around the pressure nodes of the waves generated by the acoustic field generator 100a. In other words, each of the first 110 and second 111 aggregates is positioned at a different pressure node at the end of step b). The acoustic field generator 100a may operate at a frequency allowing the formation of a standing wave comprising two nodes along the thickness of the channel 2. In a variant, the acoustic field generator 100a does not produce a standing wave along the thickness of the channel 2, but is able to create an acoustic force field that enables formation of aggregates.

(33) The expression “acoustic levitation” is employed when acoustic manipulation seeks to place objects in an equilibrium position against gravity. The equilibrium position depends on the acoustic properties of the objects and the suspending liquid, the acoustic power and the position and number of nodes of the acoustic waves. When particles or aggregates of the same or different species are in different equilibrium positions (levitating) in the channel, the expression “acoustic multi-levitation” is employed.

(34) The liquid L may be flowing during the method according to the invention and the formation of the first 110 and second 111 aggregates at step b) may be done without a stop-flow period.

(35) The acoustic focusing opposes the hydrodynamic shear induced diffusion mentioned above.

(36) Step b) may have a duration comprised between 0.1 s to 5 minutes.

(37) In other variants not shown, the methods according to the invention enable the formation of multilayer aggregates comprising three or more layers.

(38) As explained above, an aggregate of objects may be more compact than a layer of objects. FIG. 2 shows an upper view of the first aggregate 110 obtained at the end of step b) according to the invention. The first aggregate 110 comprises a set of objects O that are in contact with each other, e.g. at least 50% of the objects O constituting said aggregate 110 can be in contact with each other.

(39) The invention may enable the formation of 2D and/or 3D aggregates, e.g. depending on the strength of the acoustic field and the hydrodynamic parameters used. The definition of such 2D and 3D aggregates is given below.

(40) Further, the first aggregate 110 comprises a succession 110.sub.1 of objects O when moving along the Y axis which corresponds to a displacement along the width of the channel 2. The first aggregate 110 also comprises a succession of objects O when moving along the thickness of the channel 2. The first aggregate 110 is thus a 3D-aggregate.

(41) In an embodiment, the aggregate comprises a succession of objects when moving along the width of the channel but has a thickness formed of at least one object. In this case, the aggregate is a 2D-aggregate.

(42) Turning back to FIG. 1, an example of step c) according to the invention will now be detailed. In FIG. 1, the first 110 and second 111 aggregates are brought into contact to form a multilayer aggregate 120 of objects O. Said multilayer aggregate 120 is formed by submitting the first 110 and second 111 aggregates to acoustic waves inducing displacement of said first 110 and second 111 aggregates toward each other.

(43) These acoustic waves are generated by an acoustic field generator 100b which may operate at a frequency allowing the formation of a standing wave comprising one node along the thickness of the channel 2. In a variant, the acoustic field generator 100b does not produce a standing wave along the thickness of the channel 2 but is able to create an acoustic force field that enables formation of the multilayer aggregate.

(44) In the example shown in FIG. 1, step c) comprises submitting the aggregates 110 and 111 to acoustic waves having less acoustic nodes than the acoustic waves applied at step b). Further, in the example shown, the acoustic node formed at step c) has a transverse position which is different from the transverse positions of the acoustic nodes of the acoustic waves applied at step b).

(45) The aggregates 110 and 111 may thus pass through an area where the number of nodes decreases, leading to the refocusing of the formed aggregates, thereby generating the multilayer aggregate 120.

(46) Step c) may have a duration comprised between 10 seconds and 60 minutes.

(47) As shown in FIG. 3, a layer of a gel 101 acting as an acoustic impedance adapter may be present between the acoustic field generator 100a and the top wall 4 of the channel 2.

(48) FIG. 4 shows the evacuation of the multilayer aggregate 120 formed in the method illustrated in FIG. 1.

(49) In the embodiment shown in FIG. 4, the channel 2 is in fluidic communication with a plurality of outlets 8. The channel 2 may, as shown, be connected to three outlets 8, two of these outlets 8 may face each other and one may open out, one into the bottom wall 3 of the channel 2, and the other into the top wall 4, as shown in FIG. 4.

(50) The formed multilayer aggregate 120 may be discharged through the central outlet, this can be extended to a multilayer aggregate of 3 layers or more.

(51) Another embodiment of a method according to the invention is illustrated in FIG. 5. In this figure, first 110 and second 111 aggregates are formed at the end of step b) e.g. in the same manner as in FIG. 1.

(52) However, in this case, first 110 and second 111 aggregates are brought into contact to form the multilayer aggregate 120 by submitting said first 110 and second 111 aggregates to gravity in the absence of acoustic waves.

(53) In this example, the first 110 and second 111 aggregates may enter a zone 48 wherein the intensity of the acoustic field is reduced, in particular where there is no acoustic field. Said aggregates 110 and 111 may thus be allowed to settle to form the multilayer aggregate 120.

(54) In a variant, the first and second aggregates 110 and 111 at step b) may be formed after a stop-flow period and then the frequency and/or amplitude of the acoustic waves can be tuned in order to allow aggregates 110 and 111 settle for generating thickness and composition-controlled multilayer aggregate 120.

(55) In the example shown in FIG. 6, first and second aggregates 110 and 111 are formed as detailed in the embodiment shown in FIG. 1. However, the bottom wall 3 of the channel 2 has a varying thickness. In this example, the variation in the thickness of the bottom wall 3 may allow the acoustic waves generated by the acoustic field generator 100b to have a node or an antinode which is located on the bottom wall 3. In this case, the first 110 and second 111 aggregates may be displaced, during step c), towards the bottom wall 3 of the channel 2 and, as shown, the second aggregate 111 may be put into contact with said bottom wall 3.

(56) The formed multilayer aggregate 120 will be located on the bottom wall 3 of the channel 2.

(57) FIG. 7 shows another embodiment wherein the thickness t of the channel 2 decreases over at least a portion of its length in such a manner that the acoustic waves applied at step c) have fewer acoustic nodes than the acoustic waves applied at step b). In the example shown, the first 100a and second 100b acoustic field generators emit acoustic waves at steps b) and c) which have substantially the same main frequency.

(58) In a variant not shown, the first and second acoustic field generators emit acoustic waves at steps b) and c) which have a main frequency that is different. The difference between the main frequencies may enable creation at step c) of acoustic waves having a different number of, optionally less, acoustic nodes than the acoustic waves applied at step b).

(59) In a variant not-shown, the thickness of the channel increases over at least a portion of its length in such a manner that the acoustic waves applied at step c) have a different number of acoustic nodes than the acoustic waves applied at step b).

(60) In a variant not-shown, the aggregates can be brought into contact to form said multilayer aggregate of objects by increasing the number of nodes/antinodes.

(61) FIG. 8 shows a possible configuration to remove the multilayer aggregates 120 formed along the channel 2.

(62) A series of multilayer aggregates 120 can be formed along the channel 2 and the flow can then be stopped. A series of wells 200 placed at the opposing wall 3 to the first 100a and second 100b acoustic field generators can be used for receiving the multilayer aggregates 120.

(63) The formed multilayer aggregates 120 are then transported to the wells 200 using slow flow. This disposal system of multilayer aggregates 120 can be produced in series.

(64) FIG. 9 shows an embodiment of channel 2 observed from above which has the shape of circles 210 connected by channels 220 as shown in FIG. 9. In the latter configuration, the acoustic field generators can be cylindrical and a mosaic of aggregates can be generated.

Examples

(65) Experimental results obtained by the methods according to the invention are hereunder detailed.

(66) FIG. 10 shows a cancer cell 2D aggregate in levitation obtained in a channel of 250 μm thickness.

(67) FIGS. 11 A and 11 B show the multilevitation of particles. Particles of two species: latex and silica particles both 10 μm diameter are in equilibrium at two different positions in the thickness (250 μm) of the channel. FIG. 11A shows these particles at the end of step b) according to the invention and FIG. 11B shows these particles at the end of step c) according to the invention.

(68) FIG. 12A shows two 2D layers of 10 μm latex (dark) and silica (bright) particles forming a levitating bi-layer.

(69) FIG. 12 B shows a bi-layer composed of a 3D latex particles aggregate and a silica particle aggregate.

REFERENCES

(70) [1] Hoyos et al. Fluidic separation device: US2008/0067128 A1, [2] Callens, et al. (2008) Analytical Chemistry 80, 4866-4875, [3] Williams et al. (2008) Analytical Chemistry 80, 7105-7115, [4] Kuznetsova et al. (2009) Biotechnology Progress 25(3), 834-841.

(71) The expression “comprising a/one” should be understood as “comprising at least one”.

(72) The expression “of between” should be understood with the end points included.