IMPROVEMENTS TO APPARATUS AND METHODS FOR MANIPULATING MICRODROPLETS

Abstract

A method of handling an adherent cell in a microdroplet assaying system by conjugating an adherent cell to a microbead is provided. The method 50 comprises the steps of: loading a first plurality of microdroplets into a microfluidic space, wherein each of the first microdroplet 5 contains a microbead 52 and a first fluid; loading a second plurality of microdroplets into the microfluidic space, wherein each of the second microdroplet contains an adherent cell and a second fluid 54; merging the first plurality of microdroplets and the second plurality of microdroplets to form a plurality of merged microdroplets 56, each merged microdroplets containing the first and second fluids, at least one microbead and at least one adherent cell; and10 agitating each of the merged microdroplets 58 to cause the first and second fluids in each of the merged microdroplets to move such that at least one adherent cell adhere to the at least one microbead. [FIG. 1] 15

Claims

1-19. (canceled)

20. A method of handling an adherent cell in a microdroplet assaying system by conjugating an adherent cell to a microbead, the method comprising: loading a first plurality of microdroplets into a microfluidic space, wherein each of the first microdroplet contains a microbead and a first fluid; loading a second plurality of microdroplets into the microfluidic space, wherein each of the second microdroplet contains an adherent cell and a second fluid; merging the first plurality of microdroplets and the second plurality of microdroplets to form a plurality of merged microdroplets, each merged microdroplets containing the first and second fluids, at least one microbead and at least one adherent cell; and agitating each of the merged microdroplets to cause the first and second fluids in each of the merged microdroplets to move such that at least one adherent cell adheres to at least one microbead.

21. The method according to claim 20, further comprising the step of performing a selection, assaying, culturing or recovery process on the at least one adherent cell adhered to the at least one microbead.

22. The method according to claim 20, wherein the microfluidic space is part of a microfluidic chip configured to manipulate the first and second plurality of microdroplets via optically mediated electrowetting (oEWOD).

23. The method according to claim 20, wherein the microbead has a surface functionalisation of a polypeptide configured to facilitate cell adhesion.

24. The method according to claim 23, wherein the peptide surface functionalisation comprises one or more of the following sequences; Gly-Arg-Gly-Asp-Ser (GRGDS), Arg-Gly-Asp (RGD) or Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP).

25. The method according to claim 20, wherein the adherent cell is in its native adherent state.

26. The method according to claim 20, wherein the method further comprises the step of inspecting at least a subset of the first plurality and the second plurality of microdroplets, prior to merging, to determine the contents of the microdroplets and the number of beads or cells per microdroplet.

27. The method according to claim 26, wherein the method further comprises the step of sorting operation configured to discard one or more microdroplets except for those having a desired cell count.

28. The method according to claim 26, wherein the method further comprises the step of sorting operation to discard one or more microdroplets except for those having a desired bead count.

29. The method according to claim 20, wherein the method further comprises the step of identifying a selection of microdroplets having a bead count below a predetermined threshold and merging two or more of the selected microdroplets to increase the bead count.

30. The method according to claim 20, wherein the method further comprise the step of identifying a selection of microdroplets having a bead count at a pre-determined threshold level and splitting two or more of the selected microdroplets to decrease the bead count.

31. The method according to claim 20, wherein each of the first and/or second plurality of microdroplets further comprise a coupling promoter.

32. The method according to claim 20, wherein the first fluid and/or second fluid comprises cell growth media and the method further comprises the step of introducing a carrier phase to the microfluidic space, the carrier phase having been equilibrated with cell growth media, and wherein the carrier phase is configured to replace cell growth media depleted from the merged microdroplets.

33. The method according to claim 32, wherein the carrier phase comprises a release agent for releasing at least one adherent cell from at least one microbead.

34. The method according to claim 33, wherein the release agent is one or more of the following; trypsin, EDTA, protease or citric acid.

35. The method according to claim 20, wherein the method further comprises the step of incubating the plurality of merged microdroplets and monitoring the cell adhering to the microbead in each of the merged microdroplets.

36. The method according to claim 20, wherein the method further comprises the step of performing an on-chip reporter assay on the merged microdroplets.

37. The method according to claim 20, wherein the method further comprises the step of dispensing the merged microdroplets into a receptacle.

38. The method according to claim 37, wherein the method further comprises the step of depositing the plurality of merged microdroplets onto the surface of the treated well plate, wherein each merged microdroplets containing at least one adhered cell to at least one microbead.

Description

[0094] The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawings, in which:

[0095] FIG. 1 provides a flowchart showing the method of the present invention;

[0096] FIG. 2 shows a schematic of microdroplets containing microbeads and microdroplets containing adherent cells;

[0097] FIG. 3 shows an example configuration for carrying out the method of the present invention on a microfluidic chip;

[0098] FIG. 4 showing a single cell adhered to a single microbead;

[0099] FIG. 5 showing multiple microbeads and multiple cells in a microdroplet;

[0100] FIG. 6 showing a single microbead and multiple cells in a microdroplet;

[0101] FIGS. 7A and 7B illustrates cell viability after binding with microbeads at 4 and 22 hours, respectively; and

[0102] FIGS. 8A and 8B show cell proliferation on microbeads at 4 hours and 22 hours, respectively.

[0103] Referring to FIGS. 1 and 2, there is shown and illustrated a method of handling cells 50 such as adherent cells in a microdroplet assaying system. The method comprises conjugating adherent cells to microbeads. Referring to FIG. 1, a first plurality of microdroplets containing microbeads and a first fluid are loaded into a microfluidic space 52. The microfluidic space is part of a microfluidic chip configured to manipulate microdroplets via optically mediated electrowetting (oEWOD). A second plurality of microdroplets containing adherent cells and a second fluid are also be loaded into a microfluidic space 54. The second microdroplets can be loaded onto the same microfluidic chip as the first microdroplets, and can be positioned adjacent to the first microdroplets containing the adhered cells. The first and second plurality of microdroplets can be loaded into the microfluidic space via capillary action or it can be loaded into the microfluidic space via pressure driven flow. In some instances, a pump or a syringe may be used to load the first and/or second plurality of microdroplets into the microfluidic space.

[0104] In the next step, the first plurality of microdroplets merges with the second plurality of microdroplets 56. The second microdroplets may, for example, be caused to pair up with the first microdroplets in two paired microdroplet arrays. The second microdroplets are then caused to merge with the first microdroplets to form a plurality of merged microdroplets. Subsequently, each merged microdroplet contains the first and second fluids, at least one microbead and at least one adherent cell.

[0105] As shown FIG. 1, the method involves the step of agitating the merged microdroplets 58 via by stirring or by shaking or by any other force that is suitable to cause the first and second fluids to move such that at least one adherent cell moves towards at least one microbead and adheres itself to the microbead. Preferably, the merged microdroplets are stirred until the cells and beads come into contact and adhesion starts. The microdroplet of interest is then incubated and the process of cells adhering to the microbead is monitored.

[0106] The microbead may have a diameter of between 2 .Math.m to 200 .Math.m, but it may be more than 10, 20, 40, 80, 100, 120, 140, 160 or 180 .Math.m. In some embodiments, the diameter of the microbead may be less than 200, 180, 160, 140, 120, 100, 80, 40, 20 or 10 .Math.m. In some instances, two or more microbeads may come together to form bead clusters. The formation of bead clusters may occur by changing media conditions such as changing the pH and/or salt content of the media. Beads can cluster together to form a larger surface area such that it provides a bigger area for more adherent cells to adhere to.

[0107] Additionally, a surface of the microbeads can be coated and/or functionalised with a protein such as a short polypeptide. Examples of polypeptides sequence may include, but is not limited to, GRGD, RGD, GRGDS, or GRGDSP. The microbead can be partially or fully coated with a polypeptide. The polypeptide attached to the surface of the microbead can facilitate cell adhesion. Additionally or alternatively, the surface of the microbead can be coated with one or more of the following materials i.e. collagen, laminin and/or polystyrene.

[0108] The material of the microbead may be made from one or more of the following materials: Silica, polystyrene, latex, polyester, PMMA, magnetite and/or ferrite.

[0109] In one example, microbeads may be prepared with a surface functionalisation of a short peptide such as Gly-Arg-Gly-Asp-Ser (GRGDS). The short peptide i.e. Gly-Arg-Gly-Asp-Ser (GRGDS) can be aliquoted at 100 ug/mL in coupling buffer (Coupling buffer: 0.1 M MES, 0.5 M NaCl, pH 5.5). EDC can be immediately poured into the bead slurry. The beads are then vortex and incubate at room temperature for approximately 2 hours on a rotator. Occasionally the mix can be vortexed during incubation. The beads are then washed and resuspended in 1x PBS, 0.1% tween 20 and 0.02% NaN3, pH 7.4. The procedure as outlined above provides a microbead coated with a protein sequence of GRGDS. Microbeads may be coated with other protein sequences such as GRD.

[0110] Referring to FIG. 2, there is shown a method according to the present invention. As shown in FIG. 2, beads 62 in droplets 60 are merged with cells 64 in droplets 60 on an optofluidic device. After merging, the combined droplets 66 are agitated as indicated by the arrows 67, causing the beads 62 and the cells 64 to interact physically. The clusters of beads 62 and cells 64 are incubated inside the droplets and the cells enter their adherent state. The cluster can be inspected to see that the cell morphology has changed to be characteristic of the adherent state.

[0111] Preparation of an emulsion of the microbeads requires beads to first be suspended in a solution of cell media, before they are pumped under pressure through an emulsification apparatus. The volume bead density in the initial solution must match the required bead density in the resulting emulsion and the beads must be agitated to maintain a homogenous dispersion throughout the emulsification process. The emulsification apparatus comprises a microchannel plate chip with outlet orifices at one end, and an inlet for continuous fluid at the other end. The outlet end is immersed in to a vessel of carrier phase; typically this is an oil-based carrier phase, immiscible with the bead media. Beads pumped through the plate emerge surrounded by media at the outlet orifices where the media breaks off in to droplets. The resulting emulsion of media droplets containing beads surrounded by carrier phase can then be pumped in to an optofluidic chip for use in cell-based assays.

[0112] Preparation of an emulsion of cells requires recovering cells from an off-chip culture stock which is typically maintained in flat-bottomed cell culture flasks. Cells which are culturing in their adherent state must be resuspended using trypsin or another suitable release agent. The release agent must then be deactivated or removed in order not to inhibit subsequent return to adherent state; removal can be achieved by repeated washing steps in which cells are spun down to the bottom of a vessel in a centrifuge and the supernatant replaced with media. Deactivation can be achieved by the addition of excess protein substrate to the solution containing a protease-based release reagent. In the case that a particular occupancy of cells inside each droplet is required, the input must be diluted or concentrated such that the density of cells in the input matches the required droplet occupancy. Once the cells have been suspended and are at the required density and the release reagent has been either deactivated or removed, the cells must be pumped through an emulsification apparatus as described above for the microbeads.

[0113] The resulting emulsion is then pumped on to the optofluidic chip for droplet manipulation and the formation of adherent clusters of cells and beads.

[0114] At least one merged microdroplet containing at least one adherent cell adhered to at least one microbead may be selected for further assays. Some example assays have been performed on such cultured adherent cells, such as, for example, the introduction of a fluorescent reporter dye to the cultured adherent cells. Other example assays that could be performed on the cultured adherent cells may include: the introduction of a reporter bead, the introduction of a FRET reporter, the imaging of an endogenously expressed reporter, microscopic cell morphology measurements, lysis of the cultured cells, genetic detection assays such as PCR, isothermal amplification or fluorescence in-situ hybridisation, and DNA sequencing preparation. Alternatively the detached cells can simply be flowed off-chip for further analysis.

[0115] Referring to FIG. 3, there is shown an example configuration of a microfluidic chip comprising an oEWOD stack suitable for carrying out methods according to the present invention as disclosed.

[0116] Typically, microfluidic devices for manipulating droplets may cause the droplets, for example in the presence of an immiscible carrier fluid, to travel through a microfluidic space defined by two opposed walls of a cartridge or microfluidic tubing. Embedded within one or both walls are microelectrodes covered with a dielectric layer each of which is connected to an A/C biasing circuit capable of being switched on and off rapidly at intervals to modify the electric field characteristics of the layer. This gives rise to localised directional capillary forces in the vicinity of the microelectrodes which can be used to steer the droplet along one or more predetermined pathways. Such devices are known by the acronym EWOD (Electrowetting on Dielectric) devices. A variant of this approach, in which the electrowetting forces are optically-mediated optically-mediated, is known in the art as optoelectrowetting and hereinafter the corresponding acronym oEWOD.

[0117] Microfluidic devices employing oEWOD may include a microfluidic cavity defined by first and second walls and wherein the first wall is of composite design and comprised of substrate, photoconductive and insulating (dielectric) layers. Between the photoconductive and insulating layers, there may be disposed of an array of conductive cells which are electrically isolated from one another and coupled to the photoactive layer and whose functions are to generate corresponding electrowetting electrode locations on the insulating layer. At these locations, the surface tension properties of the droplets can be modified by means of an electrowetting field. These conductive cells may then be temporarily switched on by light impinging on the photoconductive layer. This approach has the advantage that switching is made much easier and quicker although its utility is to some extent still limited by the arrangement of the electrodes. Furthermore, there is a limitation as to the speed at which droplets can be moved and the extent to which the actual droplet pathway can be varied.

[0118] The example device as shown in FIG. 3 is suitable for the manipulation of aqueous microdroplets 1 having been emulsified into a fluorocarbon oil, having a viscosity of 1 centistokes or less at 25° C. and which in their unconfined state have a diameter of less than 100 .Math.m e.g. in the range 20 to 80 .Math.m. In some embodiments, the diameter may be more than 20, 30, 40, 50, 60, 80, 100, 120, 140, 160 or 180 .Math.m. In some embodiments, the diameter may be less than 200, 180, 160, 140, 120, 100, 80, 60, 50, 30, 30 or 20 .Math.m.

[0119] The oEWOD stack of the device comprises top 2a and bottom 2b glass plates each 500 .Math.m thick coated with transparent layers of conductive Indium Tin Oxide (ITO) 3 having a thickness of 130 nm. Each of the layers of conductive Indium Tin Oxide (ITO) 3 is connected to an A/C source 4 with the ITO layer on bottom glass plate 2b being the ground. Bottom glass plate 2b is coated with a layer of amorphous silicon 5 which is 800 nm thick. Top glass plate 2a and the layer of amorphous silicon 5 are each coated with a 160 nm thick layer of high purity alumina or Hafnia 6 which are in turn coated with a monolayer of poly(3-(trimethoxysilyl)propyl methacrylate) 7 to render the surfaces of the layer of high purity alumina or Hafnia 6 hydrophobic.

[0120] Top glass plate 2a and the layer of amorphous silicon 5 are spaced 8 .Math.m apart using spacers (not shown) so that the microdroplets undergo a degree of compression when introduced into the device cavity. An image of a reflective pixelated screen, illuminated by an LED light source 8 is disposed generally beneath bottom glass plate 2b and visible light (wavelength 660 or 830 nm) at a level of 0.01 Wcm2 is emitted from each diode 9 and caused to impinge on the layer of amorphous silicon 5 by propagation in the direction of the multiple upward arrows through bottom glass plate 2b and the layer of conductive Indium Tin Oxide (ITO) 3.

[0121] At the various points of impingement, photoexcited regions of charge 10 are created in the layer of amorphous silicon 5 which induce modified liquid-solid contact angles on the layer of high purity alumina or Hafnia 6 at corresponding electrowetting locations 11. These modified properties provide the capillary force necessary to propel the microdroplets 1 from one electrowetting location 11 to another. LED light source 8 is controlled by a microprocessor 12 which determines which of the diodes 9 in the array are illuminated at any given time by preprogrammed algorithms.

[0122] Further specific details of microfluidic chips suitable for carrying out the methods of the present invention may be found in the published patent WO 2018/234445, which is herein incorporated by reference.

[0123] The device of the present invention also provides for implementing environment controls suitable for the adherent cell conditions such as: controlled temperature, regions of different flow, controlling the carrier fluid to continuously feed cultured cells a supply of nutrients, and control of the local gas concentration in the carrier fluid surrounding the cultured cells.

[0124] For example, the adherent cell culture may be located in a region of low flow and surrounded by regions of faster flow that contain and supply nutrients and chemicals to the culture to encouraging growth.

[0125] Referring to FIG. 4, there is shown a single microbead and a single adherent cell inside a merged microdroplet. Each stage is taken at approximately 0 hours, 15 minutes, 30 minutes, 4 hours to 22 hours to show adherence of the adherent cell to the microbead. As time progresses, the single microbead and the single adherent cell come together into contact. The single adherent cell adheres to the microbead within the microdroplet as shown in FIG. 4. The arrow as indicated in FIG. 4 shows proliferation of adherent cells.

[0126] Referring to FIG. 5, there is shown a merged microdroplet containing multiple microbeads and multiple adherent cells. The merged microdroplet is monitored at each stage from 0 hours, 15 minutes, 30 minutes, 4 hours and 22 hours. FIG. 5 multiple adherent cells covering the microbeads and aggregate to one or more microbeads. In some instances, cell morphology such as structure, size and/or shape may change during the incubation period with the microbead. Changes in morphology are indicative of the phenotypic state of the cells and the growth process; it can be used as an indicator of cell health and viability.

[0127] Referring to FIG. 6, there is shown a merged microdroplet containing a single bead with multiple adherent cells. The merged microdroplet of interest is monitored at each stage from 0 hours, 15 minutes, 30 minutes, 4 hours and 22 hours. FIG. 6 shows that multiple adherent cells are able to adhere to a single microbead. In some instances, cell morphology such as structure, size and/or shape may change during the incubation period with the microbead.

[0128] FIGS. 7A and 7B provide images of a microdroplet at 4 hours and 22 hours respectively, showing cell viability tests after the cells have conjugated with the microbeads. The conditions provided enable the adherent cells to conjugate with the microbeads and the cells are left to culture on the microbeads for 4 hours. The microdroplet containing adherent cells and microbeads are then extracted and dispensed onto a tissue culture treated plate as shown in FIG. 7A. The cells are then left to culture on the microbeads for up to 22 hours. After 22 hours, the microdroplet containing adherent cells and microbeads are then extracted and dispensed onto a tissue culture treated plate as shown in FIG. 7B.

[0129] The results shown in FIG. 8A, which provides an image of a merged microdroplet at 4 hours and in FIG. 8B, which provides an image of the microdroplet at 22 hours, show that adherent cells on a single bead or on multiple beads are viable and able to proliferate during the incubation period.

[0130] Each droplet containing a plurality of cells and one or more microbeads may be manipulated according to the needs of particular sampling assays in any number of ways. Such manipulation may comprise altering the electrowetting conditions for the microdroplets such that the microdroplets de-wet or partially de-wet from the surface. The term “de-wet” as used herein refers to the change in contact angle between the droplet and the chip surface such that the droplet is pulled away from the surface.

[0131] Biological and/or chemical assays could be performed on the cultured cells that can include the introduction of a reporter bead, the introduction of a FRET reporter, the imaging of an endogenously expressed reporter, microscopic cell morphology measurements, lysis of the cultured cells, genetic detection assays such as PCR, isothermal amplification or fluorescence in-situ hybridisation, and DNA sequencing preparation. Alternatively the detached cells can simply be flowed off-chip for further analysis.

[0132] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0133] “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0134] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0135] It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.