WO2009010719A1

WO2009010719A1 - Method for cell manipulation

Info

Publication number: WO2009010719A1
Application number: PCT/GB2008/002355
Authority: WO
Inventors: Oscar Ces; David R. Klug; Mark Andrew Aquilla Neil; Peter Michael Pinto Lanigan; Richard H. Templer; Tanja Ninkovic
Original assignee: Imperial Innovations Limited
Priority date: 2007-07-13
Filing date: 2008-07-09
Publication date: 2009-01-22
Also published as: GB0713672D0; GB0810558D0; GB0810200D0

Abstract

The present invention relates to a method for the manipulation of a single cell comprising trapping and moving a droplet comprising an interior and exterior to bring it into contact with the cell and allowing interaction of the droplet with the cell, wherein one of the interior and exterior of the droplet is hydrophobic and the other is hydrophilic.

Description

METHOD FOR CELL MANIPULATION

The present invention relates to a method for the manipulation of a single cell.

Methods of cell manipulation and analysis involving effecting a physical or chemical action or stimulus, such as chemical digestion or mechanical force, on biological cells are known in the art and are used, for example, in cell analysis techniques. With many known techniques, this involves application of a physical or chemical stimulus to a population of cells. However, problems can arise with analyzing a cell population as a result of the fact that cells within a given cell population will be heterogeneous. This has prompted the need for alternative ways of analysing cells and more particularly for techniques allowing analysis and manipulation of single cells.

Single cell analytic methods are believed to overcome disadvantages associated with population averaged analytic methods. One technique known in the art involves contacting cells with solid beads which may have been coated with chemically active coatings. In particular, such methods allow more accurate information to be obtained and thus allow a better understanding of cell responses to therapeutic interventions and disease states. Single cell analysis has the potential to become an important tool for aiding and enabling the development of predictive and preventative medicine tailored to individual patients.

Microfluidic systems have previously been used to perform single cell analysis. Generally, microfluidic analysis has concentrated on the intracellular components of the cell with no sub-cellular specificity. Known analytical methods require the location of a cell in a separation channel, lysis of the entire cell, separation of the cellular components and analysis of the intracellular component of interest by capillary electrophoresis.

Such techniques necessarily involve cell destruction. Furthermore, there are limits to the extent of information that can be gained by analysis of intracellular components of a cell, particularly when considering interactions of a cell with its surrounding environment, generally mediated by the cell plasma membrane. With regard to the isolation of membrane proteins, the conventional method for isolating membrane proteins is to break open and fragment all of the membranes of a cell culture, and then to use a series of increasingly specific purification steps to finish with a single protein species. Difficulties associated with cell membrane proteomics stem to a large extent from the extreme variation in membrane protein solubility properties. The usual strategy is to try to solubilise as much of the membrane as possible and then separate it, however when this is done, the majority of proteins identified by mass spectrometry for example are not membrane proteins at all, with typically only 10-20% of the proteins being identifiable as membrane proteins.

WO 2006/059109 describes a method for analysing the plasma membrane of a single cell using a microfluidic cell analyser. The microfluidic cell analyser comprises one or more single-cell traps, a manipulator arranged to manipulate the outer surface of a trapped cell, a detection zone in communication with the single-cell trap and a detector. Manipulation of the outer surface of the cell can be achieved by exposing the trapped cell to one or more of hormones, proteins, enzymes, lipids, detergents, chemical reagents, sonication, and/or physical agitation.

Trapping of a single cell can be achieved by optical trapping within an optical cell trap. The term "optical trapping" refers to the process whereby objects of a high dielectric constant are naturally attracted to regions of high electric field, for example the maximum electric field produced in the focus of a laser beam. Alternatively optical trapping is affected by forming a dark spot in the centre of the focused beam to trap objects (with appropriate dielectric properties) which are repelled by the field, wherein the object is forced into the dark centre. The exact form of the beam focus e.g. to form lines or curves, can be used to trap extended or non-spherical cells or objects. In addition to its use for trapping cells, optical trapping has been used to trap and manipulate hexane droplets (Ward et. al. Chem Comm, 2006, 4515-1517). A small droplet of immiscible liquid, such as hexane droplets in an AOT-hexane-brine emulsion can be optically trapped by focussing a laser beam to a small, high intensity region using a microscope. The refractive index contrast afforded by the difference in refractive indices of water and hexane means that the hexane droplet is attracted to and held in the high intensity focus of the laser beam. Manipulating the optical beam then allows the droplet to be manipulated, for instance to move its position in space or to change its shape.

In addition, studies have demonstrated that the plasma membrane of a cell can be manipulated by physical contact with a polystyrene bead (Raucher et al, Biophys. J. 77:1992-2002 (1999) and Li et al, Biophys. J. 82:1386-1395 (2002)). In these studies, plasma membrane-attached polystyrene beads were pulled away from a cell by use of optical tweezers, resulting in the formation of a membrane tube or tether. These studies were used to characterize mechanical properties of a plasma membrane. The nature of cell manipulation which can be carried out by contacting a cell with a polystyrene bead is inherently limited by the physical characteristics of a polystyrene bead, such as a fixed surface area and volume, restricting the possibility of a bead being used for either the uptake of components from a cell or delivery of material to a cell.

There is a need in the art for a cell manipulation technique that allows for controlled manipulation of internal or external features of a cell, wherein manipulation can be carried out with or without loss of cell viability. A method that would allow controlled spatially specific removal of components from or delivery of material to a single cell without loss of cell viability would have huge potential for use in cell analysis studies, as a drug delivery research tool or for providing controlled dose exposure in toxicology studies. The inventors have developed a method for the controlled manipulation of a cell which involves contacting the cell with a droplet isolated from an emulsion, wherein the droplet comprises an interior and an exterior, wherein one of the interior and exterior is hydrophobic and the other is hydrophilic. This method overcomes limitations of known cell analysis techniques such as inapplicability to single cells, the requirement for cell destruction and the inability to effect spatially specific removal or delivery of a material to a cell.

Therefore, the first aspect of the invention provides a method for manipulating a cell comprising trapping and moving a droplet to bring it into contact with a cell and allowing interaction of the droplet with the cell, wherein the droplet comprises an interior and an exterior, wherein one of the interior and exterior is hydrophobic and the other is hydrophilic. Preferably, the method comprises trapping or immobilising the cell prior to and during contact of the droplet with the cell. A cell to be manipulated using the method of the invention can be trapped or immobilised to allow control or maintenance of its spatial position whilst manipulation is performed. Trapping of the cell can be achieved using a variety of physical means including optical trapping, aspiration based methods (e.g. micropipette aspiration) or dielectrophoresis. Alternatively, a cell can be immobilised, for example by adherence of the cell to a surface or to another cell.

In the context of the present invention, manipulation of a cell is taken to mean the application of a physical, chemical or biological action on a cell. Examples of manipulations that can be achieved by the method of the invention include spatially specific removal of material from a cell (examples include a cell component such as an organelle, specified fraction of a plasma membrane, a membrane protein or protein complex); induction of a cell stimulus; chemical, biological or physical disruption of a cell component; and delivery of a reagent to a pre-defined location on a cell surface or to a pre-defined intracellular target.

The method of the invention may comprise manipulation of a single cell by contacting the cell in a controlled manner with one or more droplets. Alternatively, the method may involve manipulation of two or more cells by controlled contact of each cell with one or more droplets.

In a preferred embodiment, the droplet comprises a fluid core and a coating.

In a preferred embodiment, the fluid core of the droplet comprises a water immiscible liquid. Preferably, the immiscible liquid has a refractive index that differs from the refractive index of water. The refractive index contrast afforded by the difference in refractive indices allows optical trapping of the droplet. Preferably, the water immiscible liquid is an oil, preferably an alkane, for example hexane, heptane, hexadecane or cyclohexane.

In a preferred embodiment, the fluid core comprises a water immiscible liquid. In an alternative preferred embodiment, the fluid core comprises a mixture of two or more water immiscible liquids. Advantageously, the use of two liquids in the core allows the composition of the core to be tuned, for example to aid solubility of a cargo molecule within the fluid core and fine-tune the refractive index of the droplet. The latter advantage will mean that the power of the laser used in optical trapping to trap the droplet can be varied. This will help to minimize adverse effects such as cell damage.

In an alternative embodiment, the fluid core comprises an aqueous liquid, preferably an ionic liquid, more preferably a room temperature ionic liquid (RTIL) (for example methyl imidazole with a hexafluorophosphate or tetrafluoroborate anion).

In a preferred embodiment, the coating comprises an amphiphile. If the core of the droplet comprises a hydrophobic (water immiscible) liquid, the hydrophobic portion of the amphiphile associates with the core and the hydrophilic portion of the amphiphile provides the droplet with a hydrophilic exterior. Conversely, if the core comprises an aqueous liquid, the hydrophilic portion of the amphiphile associates with the core and the hydrophobic portion provides the droplet with a hydrophobic exterior.

Preferably, the coating is a monolayer coating comprising an amphiphile. The amphiphile may be any molecule having amphiphilic characteristics, for example a lipid derivative such as a phosphatidylethanolamine (e.g dioleoyl- phosphatidylethanolamine), a phosphatidylcholine (e.g. 1, 2-deheptanoyl-sn-Glycero- 3-phosphocholine or l,2-dioleoyl-sn-glycero-3-phosphocholine), or a surfactant such as octyl-glucoside, sodium dodecyl sulphate (SDS), triton X-100, CHAPS or Zwittergent 3-12. The coating may comprise a mixture of amphiphiles, for example a phosphatidylethanolamine and phosphatidylcholine mixture. Preferably, the coating comprises a mixture of 1,2-dioleoyl -phosphatidylethanolamine (DOPE) and 1,2- diolecyl-sn-glycero-3-phosphocholine (DOPC). Coatings consisting of mixtures allow fine tuning of the properties of the droplet, such as fuginicity, fluidity, stability in-vivo, ability to dissolve or fuse with plasma membranes etc. Amphiphilic molecules making up the coating may be fluorescently labelled. In a preferred embodiment, the coating comprises a plurality of monolayers and/or bilayers, for example allowing the size of the droplet to be tailored and providing alternating layers of different properties. These monolayers and/or bilayers may be symmetric or asymmetric.

In a preferred embodiment, the coating comprises a biocompatible molecule, for example PEG (polyethyleneglycol) or a PEG labelled lipid. A coating comprising a biocompatible molecule acts as an interaction or stealth layer to prevent a cell system recognising the droplet as foreign and thereby increasing the lifetime of the droplet within a cell system. This is of particular use where the method of the invention is used for drug delivery to a cell in-vivo. The stealth layer may be a monolayer coating around but not necessarily adjacent to the fluid core or an additional bilayer in a multi-lamellar droplet (which may include molecules other than the biocompatible molecule). In alternative embodiment, a stealth layer may be absent so as to maximise interaction between the droplet and the cell. This is advantageous when the cell manipulation comprises removal of material from the plasma membrane of a cell.

Preferably, the maximum diameter of the droplet is no more than 50 microns. More preferably, the diameter of the droplet is in the range of 50nm to 5 microns, such as, in the range of 1 to 5 microns. Preferably, the lower limit for the diameter is in the order of hundreds of nanometers (e.g. 100-900 nm). Even more preferably, the diameter is in the range of 0.5-5 microns.

In an alternative embodiment, the droplet is formed from an amphiphile, preferably a long chain amphiphile. A droplet formed from an amphiphile has a micelle-like structure, with the hydrophobic portion of the amphiphile forming a hydrophobic interior and the hydrophilic portion of the amphiphile forming a hydrophilic exterior. Preferably, the hydrophobic portion of the amphiphile comprises a hydrocarbon moiety such as a long chain of the form CH₃(CH₂)_I1 wherein n is sufficiently large to drive the formation of micellar moieties and a hydrophilic group which may be a charged group (such as a carboxylate, sulphate, sulphonate, phosphate or amine group) or a polar, uncharged group (such as diacyl glycerol). As is known in the art, the length of hydrocarbon chain necessary to drive formation of micellar moieties varies depending on the nature of the hydrophilic head group of the amphiphile. Thus, it will be appreciated that n should be sufficiently large that the hydrophobic effect dominates.

Preferably, the droplet is trapped prior to being moved to contact the cell. Preferably, trapping and movement of the droplet are controlled by use of one or more of optical trapping, an aspiration based method (e.g. micropipette aspiration) or dielectrophoresis. More preferably, trapping and movement of the droplet is controlled by use of optical trapping.

In a preferred embodiment, the method comprises the step of providing an emulsion comprising a plurality of droplets, from which a droplet is trapped and moved into contact with a cell. Preferably, the emulsion is generated by mixing water (or an aqueous solution such as brine), a water immiscible liquid as described above and an amphiphile or mixture of amphiphiles as described above and agitating the mixture (for example by shaking or sonication) such that the hydrophobic effect drives generation of an emulsion comprising amphiphile coated droplets. The emulsion is preferably a microemulsion containing a population of droplets having an average diameter of no more than 50 microns. More preferably, the average diameter of the droplets is in the range of 50nm to 5 microns, even more preferably, the average diameter is in the range of 0.5-5 microns. In a preferred embodiment, a single droplet or multiple droplets are isolated from the emulsion prior to bringing them into contact with a cell. Isolation of a droplet can be carried out in a number of ways including optical trapping (if necessary followed by transfer to an isolation chamber within a microfluidic network), dilution of the solution and micropipette aspiration.

In a preferred embodiment, the emulsion is formed from 65vol% oil (preferably heptane), 25vol% water and 10vol% surfactant (preferably Triton-X). Preferably, this emulsion is diluted with additional water (or_> aqueous solvent) before a droplet is trapped therefrom. This emulsion comprises surfactant coated droplets that are particularly suitable for use in a method of cell manipulation, wherein the manipulation comprises partial solubilisation of a cell membrane.

In another preferred embodiment, the emulsion is formed from an oil (preferably hexadecane):water mixture with a ratio, by volume, of 2:5 and a DOPE:DOPC mixture with a molar ratio of 3:1. Preferably, the DOPErDOPC mixture is provided within the emulsion at a concentration of 0.7-0.8 mg/ml, preferably 0.71 mg/ml.

Preferably, the emulsion is formed by mixing components as described above and then further diluted by water or an aqueous solution, e.g. PBS, prior to use.

In a preferred embodiment, the method of the invention is a microfluidic method, comprising at least one step carried out in a microfluidic format. Preferably, the method is performed using a microfluidic assembly comprising a chamber and a microfluidic channel, wherein the method comprises introducing a flow of an emulsion comprising a plurality of droplets into the microfluidic channel, trapping one or more droplets from within this flow and moving the one or more droplets away from the flow to the chamber where it can be brought into contact with a cell within the chamber or where it can be stored for later being brought into contact with a cell. Preferably, the method comprises the step of diluting the emulsion with water or a buffer such as PBS prior to introduction into the microfluidic channel. In some preferred embodiments, the flow of the emulsion is a sheath flow within the microfluidic channel. Preferably, the chamber contains a cell culture. As used herein, a sheath flow of an emulsion refers to a focussed flow of an emulsion as a single stream surrounded by a sheath of another fluid, generally water or a buffer.

It will be appreciated that a channel is considered a microfluidic channel if at least one of the its width or depth has a maximum dimension of no more than lOOOμm. Preferably, the channel has a maximum width and/or depth of 500μm, more preferably the channel has a width and/or depth between 50 and 150μm (for example lOOμm or less).

The method of the invention enables movement of the droplet to be controlled so as to bring the droplet into contact with the cell at a pre-determined location on the cell surface. The droplet is brought into contact with the cell and allowed to interact therewith, whilst the spatial location of both the cell and the droplet is maintained and the contact time controlled. In a preferred embodiment, droplet-cell interactions are monitored using a combination of brightfield and fluorescence microscopies. This may include amongst other imaging modalities, fluorescence lifetime imaging and confocal microscopy. The method of the invention advantageously allows interaction with the cell to be controlled by control of the composition of the droplet, by control of the contact time of the droplet with the cell and by control of the spatial location of the droplet with respect to the cell.

In a preferred embodiment, the droplet comprises a reagent incorporated therein. Preferably, the reagent is incorporated into the droplet prior to contacting the cell with the droplet, preferably by inclusion during formation of a droplet-containing emulsion. The reagent may be a small molecule, a drug molecule, a fluorescent molecule or a fluorescently labelled molecule, a biological molecule (such as a protein, an antibody, a plasmid, a cell organelle, RNA or DNA), an amphiphile or an inorganic reagent (including quantum dot moieties). It will be appreciated that the reagent may be incorporated into the fluid core, the coating or may sit on the interface of the core and the coating depending on whether the reagent is hydrophobic, hydrophilic or amphiphilic. A method of the invention which involves the use of a droplet comprising a reagent enables delivery of the reagent to a cell.

The reagent may be a labelling molecule, for example a fluorescent molecule or a reagent of interest which has been fluorescently labelled. The incorporation of a labelling molecule allows (a) the visualisation of the location of droplet, especially when trapping the droplet; (b) ascertaining whether exchange of material between the droplet and the cell has taken place; (c) if the reagent is incorporated in the fluid core, the measurement of leakage from the fluid core; (d) the location of the reagent at anytime including after delivery.

In a preferred embodiment, the invention provides a method for manipulation of a cell, wherein the manipulation comprises physical deformation of a cell membrane by contacting the surface of a cell with a droplet, maintaining contact of the droplet with the cell surface for time sufficient to allow the droplet to fuse with the cell membrane (preferably 20 seconds or less, for example 10-20 seconds) and moving the droplet away from the membrane so as to deform the membrane. Preferably, physical deformation results in the formation of a membrane tether extending between the cell and the droplet. In this embodiment, the exterior of the droplet has membrane fusagenic properties. It has been demonstrated that material from the cell (for example membrane proteins) can be transferred from the cell to the droplet via the tether. Thus, this method can be utilised as a method of removal of material from a cell. A preferred droplet for use in a method for formation of a membrane tether has a coating comprising a mixture of DOPE and DOPC, preferably comprising at least 75 mol% DOPE. Tailoring the amounts of DOPE and DOPC in the mixture allows control of the fusogenic properties of the droplet. Preferably, the droplet comprises a DOPE/DOPC coating and a hexadecane core.

Advantageously, the invention therefore provides a method which avoids solubilising the whole membrane at one time, and instead allow performance of stepwise and spatially selective sampling of the plasma membrane of a single cell under controlled conditions. This approach has the potential to greatly reduce the problem caused by high abundance proteins (including cytosolic systems) masking low abundance ones.

In another preferred embodiment, the invention provides a method for manipulation of a cell, wherein the manipulation comprises solubilization of the plasma membrane of a cell by contacting the plasma membrane with a droplet, wherein the coating of the droplet comprises for instance a surfactant. Solubilization is preferably partial solubilization, of a portion of the plasma membrane. Partial solubilization of the plasma membrane of a cell by contacting it with a surfactant-coated droplet can be used to transfer membrane-associated protein to the droplet. Preferably, the droplet comprises a heptane core and a coating comprising a surfactant, preferably Triton X- 100.

In another preferred embodiment, the invention provides a method for manipulation of a cell, wherein the manipulation comprises delivery of a reagent to a specific location on or within a cell by contacting the cell with a droplet comprising a reagent and allowing delivery of the reagent to the cell. The specific location on or within a cell may be a defined sub-cellular location such as targeted region of the plasma membrane. Preferably, wherein the method comprises delivery of a reagent to a location within the cell, the method comprises contacting the cell with a droplet and allowing the droplet to enter the cell. In this embodiment, the coating of the droplet preferably comprises an amphophilic molecule that is resistant to membrane fusion and/or a biocompatible molecule that prevents the droplet from being identified as foreign.

In yet another preferred embodiment, the invention provides a method for manipulation of a cell, wherein the manipulation comprises removal of material from a cell by contacting the cell with a droplet, allowing material from the cell to enter or complex with the droplet and moving the droplet away from the cell. The method may comprise cleavage of a cell surface protein by contacting the cell with a droplet having a coating incorporating a protease, a droplet having a coating comprising a surfactant as described above or a fusagenic droplet as described above

In methods where the manipulation comprises removal of material from a cell, a biological target (e.g. protein, protein complex, lipid raft/specific membrane region, cell organelle, lipid) on the cell of interest may be fluorescently labelled. This will allow the user to monitor whether the target moiety has been transferred to the droplet.

In instances where the droplet is used to effect removal of material from a cell, the droplet is analysed downstream, after movement away from the cell, using any one or more of a variety of analytical techniques including mass spectrometry, electrophoresis based methods, multi-dimensional fluorescence imaging (with respect to either one or all wavelength, lifetime and polarisation and in either two or three dimensions), labelled readout using microarray technologies or optical finger printing technologies.

It will be appreciated that as well as used to remove material from a cell, the techniques disclosed herein can be employed to remove material from a surface or to remove material that is suspended in a liquid. Therefore, in a second aspect the present invention provides a method for removal of material from a surface or from suspension within a liquid, comprising trapping and moving a droplet to bring it into contact with a surface bearing a material or a suspended material and allowing partitioning of the material into the droplet, wherein the droplet comprises an interior and an exterior, wherein one of the interior and exterior is hydrophobic and the other is hydrophilic. It will be appreciated that preferred features for the first aspect of the invention apply equally to the second aspect.

In a third aspect, the present invention provides a droplet for use in a method of cell manipulation, wherein the droplet has a maximum diameter of 50 microns and wherein the droplet comprises a fluid core and a coating and wherein the fluid core comprises a water-immiscible liquid and the coating comprises an amphiphilic lipid molecule or lipid derivative.

In a preferred embodiment, the water immiscible liquid is an oil, preferably an alkane, for example hexane, heptane, hexadecane or cyclohexane. In a preferred embodiment, the fluid core comprises a mixture of two or more water immiscible liquids.

In a preferred embodiment, the coating comprises a lipid derivative such as a phosphatidylethanolamine (e.g dioleoyl-phosphatidylethanolamine) or a phosphatidylcholine (e.g. 1, 2-deheptanoyl-sn-Glycero-3-phosphocholine or DOPC) or a mixture thereof. Preferably, the coating comprises a DOPE/DOPC mixture and a hexadecane core. Preferably, the DOPE/DOPC mixture comprises at least 75mol% DOPE.

In a preferred embodiment, the coating comprises a monolayer or a plurality of monolayers and/or bilayers which may be symmetric or asymmetric.

In a preferred embodiment, the coating comprises a biocompatible molecule, for example PEG (polyethyleneglycol) or a PEG labelled lipid.

Preferably, the diameter of the droplet is no more than 50 microns. More preferably the diameter of the droplet is in the range of 50nm to 5 microns. Preferably, the lower limit for the diameter of the droplet is in the order of hundreds of nanometers (e.g. 100-900 nm) more preferably from 0.5-5 microns.

In a preferred embodiment, the droplet comprises a reagent incorporated therein. Preferably, the reagent is a small molecule, a drug molecule, a fluorescent molecule or a fluorescently labelled molecule, a biological molecule (such as a protein, an antibody, a plasmid, a cell organelle, RNA or DNA), an amphiphile or an inorganic reagent (including quantum dot moieties).

In a fourth aspect, the present invention provides an emulsion comprising droplets for use in a method for manipulation of a cell, wherein the emulsion and droplets comprised therein are as defined in respect of the method of the first aspect of the invention. Preferably, the emulsion comprises a plurality of droplets as defined in the third aspect of the invention, dispersed within an aqueous continuous phase.

Preferably, the emulsion is provided as a stock emulsion which can be further diluted by water of an aqueous solution such as PBS prior to use.

In a fifth aspect, the present invention provides a microfluidic assembly comprising a first delivery channel for delivery of an emulsion comprising droplets as defined herein, second and third delivery channels for delivery of a solvent, a microfluidic channel having a first end at which the first, second and third delivery channels converge and a second end provided with an outlet or a storage reservoir, a chamber and a linking channel which extends between one of the second or third delivery channels, upstream from the microfluidic channel, and the chamber. Preferably, the first delivery channel is positioned between the second and third delivery channels, such that, when a solvent and an emulsion containing droplets are delivered, a sheath flow of droplets within the microfluidic channel is established. In use, a droplet in the microfluidic channel is trapped and moved against the flow therein to chamber via the linking channel. Preferably, the chamber is an analysis chamber in which a cell can be located for manipulation by contact with a droplet or a storage chamber for storage of isolated droplets prior to use. A microfluidic assembly may comprise both an analysis chamber and a storage chamber. Thus, the microfluidic assembly which is for isolation of one or more droplets allows the generation of a sheath flow comprising droplets. From this sheath flow, one or more droplets can be trapped and isolated for further use, for example in a method as described above.

In a sixth aspect, the present invention provides a microfluidic assembly comprising a first channel extending between a first inlet connected to an emulsion storage chamber, for delivery of an emulsion comprising droplets as defined herein, and second inlet for delivery of a buffer (e.g. PBS) or cell culture media, wherein the second inlet also connects the first channel to a chamber, and a dividing flow channel extending between a third inlet, for delivery of a fluid (for example a buffer such as PBS), and an outlet, wherein the dividing flow channel intersects with the first channel so as to form a microfluidic junction and wherein the first channel comprises a first portion extending between the first inlet and the microfluidic junction and a second portion extending between the microfluidic junction and the second inlet. In use, a fluid flow is established within the dividing flow channel and an emulsion flow is established in the first portion of the first channel and a flow of buffer or cell culture media is established within the second portion of the first channel. The emulsion flow, the fluid flow and the buffer/cell culture media flow meet at the microfluidic junction where the emulsion flow is diverted from the first channel, into the dividing flow channel, thereby isolating the emulsion flow from the chamber. A droplet from the emulsion flow can be trapped at the microfluidic junction and moved across the dividing flow channel flow therein to the cell culture chamber via the second portion of the first channel (which acts as a linking channel between the emulsion flow and the chamber). Preferably, the chamber is an analysis chamber in which a cell can be located for manipulation by contact with a droplet or a storage chamber for storage of isolated droplets prior to use. A microfluidic assembly may comprise both an analysis chamber and a storage chamber. From this microfluidic junction, one or more droplets can be trapped and isolated for further use, for example in a method as described above.

In a seventh aspect, the present invention provides a microfluidic method for the isolation of a droplet as defined herein, the method comprising providing a flow of an emulsion comprising a plurality of droplets within a channel in a microfluidic assembly that also comprises a chamber, trapping (preferably by optical trapping) one or more of the droplets from the emulsion flow and moving the one or droplets away from the emulsion flow, via a linking channel, to the chamber. Preferably, the emulsion flow is a sheath flow and the trapped droplets are moved against the direction of the sheath flow to a linking channel positioned upstream of the sheath flow. Preferably, the microfluidic assembly is as defined in the fifth or sixth aspects of the invention. All preferred features of the invention apply to all other aspects mutatis mutandis. In particular, it should be understood that the features of the droplets and the emulsion described in respect of the first aspect of the invention are applicable to the droplets and the emulsion when described in respect of other aspects of the invention.

The invention may be put into practice in various ways and a number of specific embodiments will be described by way of example to illustrate the invention with reference to the accompanying drawings in which:

Figure 1 shows a schematic view of generation of a droplet.

Figure 2 shows a schematic view of a microfluidic assembly of the present invention.

Figure 3 shows an exploded view of a microfluidic assembly of the present invention.

Figure 4 shows a schematic view of an alternative microfluidic assembly of the invention.

Figure 5 shows an exploded view of the assembly illustrated in figure 4.

Figure 6 shows generation of a membrane tether between a cell and a droplet.

Figure 7 shows a fluorescent micrograph of a droplet after contact with a fluorescently labelled cell.

Figure 8 shows the trapping of droplets in a microfluidic assembly as illustrated in figure 2.

Figure 9 shows the trapping of droplets in a microfluidic assembly as illustrated in figure 4.

Figure 10 shows a schematic of trapping microscope used in experiments with traps controlled by a Lab view point click interface, in which Il = trapping laser input, Bl = polarising beam splitter, M 1-2 = IR 1064nm dielectric mirrors, λ/4 = quarter wave plates, A 1-4 = computer controlled stepper motors, Ll = focal length = 100mm planovex IR AR coated refocusing lens, L2 = focal length = 100mm planovex IR AR coated lens, SB = supporting block of microscope turret, B2 = IR trapping dichroic, B3 = FITC dichroic, 12 = Mercury lamp excitation input, Fl = fluorescence output to camera/eyepiece and Cl = bright field condenser and lamp housing.

Figure 11 shows (a) a bright field image showing droplet (arrow) prior to contact with BE cell, b-c) Fluorescence images of a droplet pulling tether from BE cell at a rate of 0.5μm/sec. Scale, nanoblob size = 2.9μm.

Figure 12 shows a) a bright field image of a droplet (rectangle) after contact with polarized human BE cell, b) simultaneously acquired bright and fluorescence image, c-e) Series of fluorescence images showing pulling of tether by droplet at 0.5μm/sec. Box indicates approximate trap location due to a small amount of hysterisis in actuator movement. Scale, size of box = 3x3μm.

Figure 13 shows a determination of the temporal stability of triton-X coated droplets with a . heptane core using a DLS platform. Size distribution of the droplets is determined after 29 minutes according to intensity (figure 13a) or volume (figure 13b).

As illustrated in figure 1, a droplet 1 having a fluid core 2 comprising an oil and an amphiphile coating is generated by mixing an oil with water 3 (or an aqueous solution such as brine). An amphiphile for forming the coating 4 of the droplet (for example phosphatidylethanolamine) is added to the mixture, together with any other reagents that are to be incorporated into the droplet coating. The resulting mixture is subjected to agitation (e.g. shaking or sonication) to form an emulsion. Typically, the oil:water:coating ratio is in the region of 12.5:25:1. Sonication of the oil-in-water mixture leads to the formation, driven by the hydrophobic effect, of an emulsion comprising oil droplets having a nm to micron size distribution, coated with an amphiphile monolayer. A droplet in which the fluid core comprises an aqueous liquid, for example an ionic liquid is produced by the method described above, but reversing the oil:water ratio.

A droplet can be formed having a monolayer, bilayer or multi-lamellar coating. Whether the coated droplet has a monolayer, bilayer or multi-lamellar coating is determined by the amount of that is amphiphile added to the oil:water mixture. It will be appreciated that the orientation of the amphiphilic molecules in the coating will be dependent on whether the fluid core comprises an oil or an aqueous liquid. If desired, further layers can be added to the coating.

Furthermore, additional reagents can be added to the oil/water/amphiphile mixture at different stages to allow incorporation into the droplet core or coating.

Due to the difference in refractive index between the oil and water, once formed the oil droplets can be captured individually using an optical trap and then moved to bring them into contact with a cell.

In a preferred embodiment, the coated droplet is brought into contact with the trapped cell using optical trapping. As^* an alternative to optical trapping, aspiration based methods or dielectrophoresis could be used. The cell and the coated droplet, once brought into contact, are allowed to interact. Depending on the properties of the droplet, this interaction can lead to direct fusion of the droplet with the cell, or chemical, biological or physical disruption of the cell or a cell component which may induce a cell stimulus, delivery of a reagent into the cell or removal of a cell component. Manipulation of the cell can be carried out with or without loss of cell viability.

The method of the invention can be used for drug delivery or as a drug delivery research tool. In addition, the method of the invention can be used to provide a cell with a controlled dose exposure to a reagent in a toxicology assay. Furthermore, the method of the invention can be used.to extract a cell component for analysis.

Chemical disruption of the plasma membrane of a trapped cell can be achieved by contacting the cell with a droplet wherein the coating of the droplet comprises a molecule that reacts or interacts with the plasma membrane. Selective solubilization of the cell can be brought about by contacting the cell, in a controlled manner, with one or more droplets having a coating comprising a surfactant such as sodium dodecyl sulphate (SDS), triton X-100, CHAPS, octylglucoside or Zwittergent 3-12. Depending on the composition of the coating, the plasma membrane of the cell can either be lysed, leading to the formation of large complexes containing protein or protein complexes, detergent and membrane lipids, or solubilized by replacement of membrane associated lipids with detergent molecules, leading to the formation of smaller individual detergent-protein complexes which are free of membrane lipids.

The solubilization effect seen is dependent on the ratio of detergent present in the coating to protein present in the cell plasma membrane, contact time and the composition of the droplet.

Droplets comprising amphiphile coatings consisting of lipid derivatives such as short chain phosphatidylcholines (e.g. 1, 2-Diheptanoyl-sn-Glycero-3-Phosphocholine) can effect membrane destabilization and can therefore can be employed for similar purposes as droplets having a coating comprising a surfactant. By integrating lipids such as phosphatidylethanolamine or lipid mixtures, droplet coatings can be made to be fusogenic so as to increase adherence to the cell skeleton. A preferred fusogenic droplet comprises a hexadecane core and a coating comprising a DOPE/DOPC mixture.

In an alternative form, the amphiphilic coating of the droplet is resistant to membrane fusion and cellular attack. A droplet with a fusion-resistant coating will be long-lived and can be moved by optical trapping to enter a cell and deliver its cargo to a designated location within the cell.

Biological disruption of a cell can be achieved by contacting a trapped cell with a droplet wherein the coating of the droplet incorporates for example a biological molecule such as a protease to cleave proteins exposed on the cell surface when the droplet is brought into contact therewith. Choice of protease incorporated within the coating and time of contact between the droplet and the cell can be controlled so as to attain the desired size of cleaved peptide fragment. Biological approaches could also be used to induce controlled cytoskeletal degradation to release anchored proteins/membrane fragments and controlled exocytosis of the plasma membrane. A coated droplet can also be loaded with receptors or other biological molecules so as to induce a signaling cascade at the point of contact of the droplet with the cell.

Physical disruption of a cell can be achieved using a droplet provided with a coating having fusogenic properties. For example, generation of a membrane tether is achieved by bringing a droplet into contact with the plasma membrane of a cell, allowing fusion to occur and then moving the droplet away from the cell (figures 5 and 10). Due to fusion of the droplet with the plasma membrane, movement of the droplet away from the cell distorts the membrane, pulling away a membrane portion in the form of a tether. Cell material can transfer, via the tether, to the droplet with the droplet acting as a reservoir for storage of extracted cell material (e.g. lipid and/or protein plasma membrane material). A membrane tether formed in this way can be dissected from the main body of the cell to permanently transfer material from the cell to the droplet using a focused detergent stream (microfluidic format) and/or a second coated droplet to "cut" the tether. Alternatively, the droplet can simply be pulled away, leading to dissection of the tether. The droplet and/or tether can then be collected in continuous flow or placed in storage before being analyzed, for example to analyse cell material transferred to the droplet. As the tether is dissected, the cell plasma membrane self-repairs and cell viability is maintained.

Thus, physical disruption of the cell membrane to form a membrane tether provides a method for removing material, such as membrane proteins, from a cell. Methods whereby an immobilised cell is brought into contact with a droplet under controlled conditions thereby facilitating transfer of material from the plasma membrane of the trapped cell to the droplets with the former retaining viability are described in detail in the following examples. Cell- droplet interactions have been monitored using a combination of brightfield and fluorescence imaging. The droplet can then be transferred downstream or stored with a view to conducting proteomic analysis. Controlled contact, both spatial and temporal between the droplets and the trapped cell is mediated by optical trapping. As further described herein, with minor modifications this technique can be used to induce a cell stimulus and delivery of a reagent to a pre-defined location on a cell surface or to a pre-defined intracellular target.

A droplet can also be used to remove material from a cell without formation of a membrane tether, by bringing a droplet into contact with a cell, allowing material to transfer between the cell and the droplet and then moving the droplet away from the cell. This has been observed experimentally by fluorescence transfer to the droplet.

The method of the invention may be performed using a microfluidic assembly. Figure 2 shows a schematic view of a microfluidic assembly, in the form of a microfluidic chip design, comprising a first inlet 20 for an emulsion comprising droplets prepared as described above and second and third inlets 21 for an aqueous solvent (e.g phosphate-buffered saline (PBS)). Each of the three inlets has a width of 50 μm. The three inlets converge to form a microfluidic channel 22 having a width of lOOμm. The inlets converge at the first end 220 of the microfluidic channel and the second end 221 of the microfluidic channel is provided with an outlet or reservoir 23. In addition, the assembly comprises a linking channel 24, linking one of the second or third inlets with a cell culture chamber 25. In use, a solvent flow is established within the microfluidic channel 22 by introduction of an aqueous solvent (e.g. PBS) via the second and third inlets 21. Once a flow is established, an emulsion containing droplets is introduced via the first inlet 20 into the microfluidic channel 22 and thereby focused in a sheath flow. The assembly allows single droplet isolation followed by movement of an isolated droplet to a chamber for interaction with a cell. A droplet of interest within the microfluidic channel is trapped 26 to ensure that it droplet remains stationary within the solvent flow. Once trapped, the droplet of interest 26 is moved against the sheath flow, to the linking channel 24 and via the linking channel into the cell culture chamber 25 where it can be brought into contact with a cell to be manipulated.

By controlling the position of the droplet relative to the sheath flow it will be possible to isolate the droplet and then move it against the direction of the sheath flow before being delivered to an isolation chamber. This isolation chamber may include the cell with which the droplet is intended to interact or may act as a reservoir within which a reduced number of droplets may be stored for later use. A microfluidic assembly of the invention may be produced in the form of a PDMS chip as illustrated in Figure 3. The assembly comprises a cover slide 30 on which, within a defined area, a cell is trapped and a PDMS chip 31 on which is formed a microfluidic arrangement as described above. The PDMS chip 31 is positioned over the cover slide 31 such that the area in which the cell is trapped corresponds to the chamber of the PDMS chip 31. In addition, the assembly comprises a microscope slide 32 which is provided with inlets and an outlet in communication with the channels of the PDMS chip 31. In use, an emulsion comprising droplets is provided to the first channel via a first inlet 33 and an aqueous solution, for example PBS, is provided to the second and third channels via second and third inlets 34. The outlet 35 enables removal of solvent from the second end of the microfluidic channel.

Figure 4 shows a schematic view of an alternative microfluidic assembly, in the form of a microfluidic chip design, comprising an emulsion storage chamber 36 and a cell culture chamber 37, linked by a first microfluidic channel 38 that has a width of 50μm. A first inlet 39 at one end of the microfluidic channel is for the introduction of an emulsion comprising droplets prepared as described above and a second inlet 40 at the other end of the channel is for introduction of an aqueous solvent (e.g phosphate- buffered saline (PBS) or cell culture media). The assembly also comprises a second channel 41, referred to as a dividing flow channel, extending between a third inlet 42 for introducing an aqueous solvent (e.g. PBS or cell culture media) and an outlet 43. The dividing flow channel intersects with the first channel at a microfluidic junction 44. Each of the three inlets has a width of 50μm, although the central channel can be wider, e.g. 100 μm. A side flow is established, for example by tailoring of the various inlet flow rates, within the first microfluidic channel, as represented by arrow 45. Once the slow side flow is established, an emulsion containing droplets is introduced via the first inlet 39 into the microfluidic junction 44 and swept into the outlet channel 43 by the inlet flows represented by arrows 45 and 46 (the central flow system).

The assembly allows single droplet isolation followed by movement of an isolated droplet to a chamber for interaction with a cell. A droplet of interest within the microfluidic channel 38 is trapped to ensure that the droplet remains stationary within the solvent flow. Once trapped, the droplet of interest 47 is moved across the microfluidic junction 44, via the second inlet 40 into the cell culture chamber 37 where it can be brought into contact with a cell to be manipulated.

This isolation chamber may include the cell with which the droplet is intended to interact or may act as a reservoir within which a reduced number of droplets may be stored for later use.

The outlet reservoir can be used as a drain for used droplets that can be sorted downstream e.g. by fluorescence sorting.

Because of the dynamic fluid environment in the microfluidic assemblies illustrated in figures 2 and 4, adaptive control based on programmable diffractive optics can be used to maintain the droplet' s position and orientation, for example in the assembly shown in figure 2, to act against the sheath flow within which the droplet is contained. In addition, by using multiple optical traps the shape of the droplets can be controlled so as produce changes in the local shape of the droplet, (see Ward et. al. Chem Coram, 2006, 4515-1517). This can be used to change the shape of the droplet from spherical so as to control the local curvature of the droplet surface which in turn can alter the chemical or physical properties of the droplet coating. In addition, the ability to manipulate both the shape and the orientation of the droplet allows matching the shape of the droplet to the surface with which it is interacting. The technique can also be used to pull a single droplet into two (or more sub units) leaving a (or multiple) thin threads between them. The coating on the droplet can be designed to stabilize the formation of such threads, which in turn can be used beneficially in the manipulation of the cell. .

In order to control the droplet-cell interactions it is important to ensure that in the cell culture chamber, the medium contains only the cell under study and the droplet with which it will interact. This will help to minimise unwanted interactions with droplets that may be otherwise floating in solution although in an alternative embodiment such interactions may be beneficial-for instance probing the accessibility of a cell surface. The assembly described above allows this isolation. Isolation of single or multiple droplets is therefore advantageous before or after interactions with cells. In some embodiments multiple droplet-cell interactions may be required and in such instances the number of trapped/isolated droplets will be varied accordingly. Isolation of the droplet can be carried out in a number of ways including optical trapping (if necessary followed by transfer to an isolation chamber within a microfluidic network), dilution of the solution and micropipette aspiration.

If so required a microfluidic assembly of the invention may comprise more than one trap for the droplets or cells, multiple reservoirs and multiple cell storage chambers and/or two or more detection zones.

The invention is further illustrated by reference to the following non-limiting examples.

Examples

A trapping arrangement that has been used to perform a cell manipulation method of the invention is described below.

The following abbreviations are used herein: DOPE, l,2-Dioleoyl-sn-Glycero-3- Phosphoethanolamine (also known as dioleoylphoshatidyethanolamine); FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate- buffered saline; NBD-PE. n-(7-nitro-2,l,3-benzoxadiazol-4-yl)-dioleoyl-sn-glycero-3- phophoethanolamine; EDTA, ethylenediamine tetraacetic acid; BE cells, a human colon carcinoma cell line; FTTC, fluorescein isothiocyanate; PDMS, polydimethyl siloxane.

Laser Trapping Arrangement and Fluorescence Imaging

Trapping experiments were conducted on a Nikon TE2000-E automated microscope, with custom-built laser trapping optics suspended from the external rear port for two beam optical manipulation as shown in figure 7. The cage plate (Linos/Thorlabs) based optical system, which extended out from the modified supporting block (SB) of the microscope objective turret allowed for compact and safe containment of the beam without the requirement for any further optics on the bench. Furthermore the compact fibre coupled laser source with diffraction limited beam quality (IPG Photonics, YLM-5, 5W, 1070nm, linearly polarised) was directly fed in to the trapping optics by a set of custom made cage plates (II).

To facilitate two trapping beams a Michelson interferometer type arrangement was constructed using a polarising beamsplitter (Bl), two quarter wave plates QJA) and two steering mirrors (Ml and M2). The linearly polarised input laser at an angle of 45 degrees was then split and recombined into two independent beams of opposite polarisation, which were manoeuvred by the mirrors independently.

Plane mirror mounts placed at each arm of the interferometer were modified for the fitting of linear actuators (Al-4) (NSA-12, Newport, 11mm range, O.lμm resolution), which allowed for precision steering of the optical traps in the x-y plane. Actuators were connected up to an 8-axis switch box and hand held controller (NSC-SB and NSC-200 respectively, Newport) and computer interfaced via a RS-485 to RS-232 converter (NSC-485-232-I, Newport) as part of the NewStep Expandable Motion Controller System (Newport). These mirrors were then imaged using a Ix telescope (Ll and L2) onto the pupil plane of the microscope objective (60x 1.2NA, water immersion, Nikon) to minimise the loss of power of the trapping beams when steering the traps in the image plane. No beam expansion was required as the output diameter of the fibre laser was 8mm, which was sufficiently large to fill the objective pupil. Refocusing of the traps was achieved by sliding the lens (Ll) in and out.

The trapping dichroic (B2) (Z900DCSP, Chroma) was located below the microscope objective and held into position by the supporting block of the microscope objective turret. Below the trapping dichroic was situated a filter cube carousel incorporating a FITC cube (B3) used for mercury lamp excitation (12) and epifluorescence imaging (Fl) of EGFP labelled cells.

Both bright field images obtained by the transillumination system (Cl) and fluorescence images were captured by a cooled digital camera (ORCA- ER, Hamamatsu) situated on the left side port (100% transmission side) of the microscope. For fast screening of cells and manoeuvring of droplets in the sample plane, a motorised x-y stage was utilised (Proscan, Prior).

Actuators were operated via an in-house Labview program (Labview 8.1, National Instruments) and allowed the traps to be positioned in the field of view of a 512x512 pixel image by a mouse click, where the location of the trap was indicated by an over laid coloured rectangle. In this case the interface was such that the trap would move sequentially along the x-axis of the image followed by a movement along the y-axis.

The speed and acceleration of the traps could be controlled by dials and sliders on the program's front panel.

Determination of temporal stability

The temporal stability of droplets was determined using a DLS platform. Figure 13 illustrates the size distribution by intensity (figure 13a) and by volume (figure 13b) of triton-X coated droplets with a heptane core. After 29 minutes, the system consists of two populations, centred around 200 nm and 4 microns respectively. This protocol allows the assessment of the stability of the droplets. It also allows an assessment of the effect of a particular, for example oil, on the coating allowing optimisation of the process. Trapping experiments were carried out using droplets centred around 4 microns.

Example 1 - Generation of a Membrane Tether with a Hexane/DOPE droplet The following procedure was carried out to demonstrate fusion of a droplet with a labeled DOPE coating with trypsinised BE cells.

DOPE NBD-PE was prepared by mixing DOPE (95.76μl at lOmg/ml concentration in chloroform) to NBD-PE (10.9μl at lmg/ml concentration in chloroform) and drying the mixture. The dried mixture was then dissolved in cyclohexane (lOOμl) and added to water (ImI). A droplet solution was produced from a mixture comprising 5μl of lmg/ml of DOPE NBD-PE in chloroform, 0.1-0.5μl hexane and ImI H₂O. This led to formation of droplets with a maximum diameter in the region of ~lμm.

BE cells were grown in 150cm² tissue culture flasks with DMEM containing 10% Foetal Calf Serum, 100IU/ml Penicillin and 100 μg/ml Streptomycin. Confluent cells were detached with ImI trypsin-EDTA solution (trypsin 5g/ml, EDTA 2g/ml) for 5 minutes. 8 ml DMEM was added. 4 ml of the resulting cell suspension was mixed with 40ml of Hanks Balanced solution. Approximately 50μl of the resulting cell solution was dropped onto a glass cover slide (CS). The position of the cell of interest in this case was fixed due to attachment of the cell to another cell. However, optical trapping could be used to achieve the same effect.

5μl of the droplet solution was added to the CS. The droplets were localized by fluorescence imaging (Nikon TE2000-E, x604 NA=I.2, water immersion objective) with a FITC filter. A droplet was then trapped with a 15OmW trapping laser (IPG Photonics, Ytterbium fibre laser at wavelength=1070nm, diffraction limited focussed laser spot size =1 micron) and moved to bring it into contact with the membrane of an optically trapped cell. Contact between the cell membrane and the droplet was maintained for ca. 10s to allow fusion to occur. The droplet 40 was moved slowly away from the cell 41, thereby pulling the fused part of cell membrane with it and leading to formation of a membrane tether 42 as observed by microscopy and shown in Figure 6.

Example 2 - Generation of a Membrane Tether with a Hexadecane/DOPE/DOPC Droplet

The following procedure was carried out to demonstrate fusion and generation of a membrane tether using lipid coated oil droplets, typically 0.5-5 microns in size and composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (1,2- dioleoyl-phosphatidylethanolamine and 1, 2-dioleoyl-sn-glycero-3-phosphocholine). These droplets were brought into contact with target BE cells using optical trapping. This facilitated controlled droplet-cell interactions which resulted in membrane tethers being formed between the two moieties across which material transfer was visualized This process, which can be termed nanodigestion, can be used for spatially selective sampling of the plasma membrane of single cells with a view to undertaking single cell proteomic assays.

An emulsion stock solution (droplet solution) was produced by mixing anhydrous frozen lipid stocks of DOPE and DOPC in a niolar ratio of 3: 1. 5 mg of this mixture was added to a clean glass vial containing 2ml of hexadecane (density = 0.773g/cm³) and 5ml of single distilled water. The mixture was then vortexed for a few seconds to form a stable cloudy oil in water emulsion that was observed to not ripen over the course of at least a week. Oils with other chain lengths (shorter or longer) e.g. heptane were found to form less stable emulsions, which separated out into water and oil phases in a few minutes. The lifetimes and temporal size distribution of DOPC/DOPE/oil droplets were measured using Dynamic light scattering (DLS) (model, Malvern Instruments). DLS can be used to measure the size and stability of any type of droplet.

Adherent BE human carcinoma cells expressing enhanced green fluorescence protein (EGFP) at the plasma membrane via a CAAX linker were cultured in an incubator at 37°C and 5% CO₂ in 25ml filter capped flasks containing phenol free media (DMEM (GibCo) with 10% Foetal Calf Serum, lOOμl/ml Penicillin and 100 μg/ml Streptomycin. These were then split when reaching 80% confluency in a culture hood by removal of culture media, washing with sterile phosphate buffered saline (PBS) (pH7.4, GibCo) and incubation with 2ml of trypsin solution (Sigma) containing trypsin 5g/ml and EDTA 2g/ml diluted to 10% in PBS for 2-3minutes. Once cells were detached, 5ml of culture media was added to the flask and the cells were gently re-suspended by pipetting up and down for 30seconds.

0.5ml of cells was then carefully pipetted onto the centre of cover glass slides before being placed in well plates. The well plates were then transferred into an incubator for 15 minutes whilst the cells attached to the glass. Cells were then removed from the incubator- and a further 2-3ml of culture media was then added gently to each side of the wells before being returned to the incubator for 24 hours in order to allow them to polarise. The cover glass slides were then transferred onto the microscope slide holder. The emulsion stock solution was diluted by a factor of 1:100 in clean PBS, and then shaken vigorously by hand and vortexed for 30 seconds, in order to redisperse the system. 5-10μl of this solution was pipetted onto the coverglass to which the polarised cells were adhered using a Gilson pipette.

The Labview program was then run and the joystick of the motorised stage used to search for appropriate droplets (sized between l-5μm) to optically trap. Once a droplet was trapped (laser powers used were typically ~200mW at the pupil plane of the objective, typically ~40-60% transmission to the sample plane at 1070nm the motorised stage was used again to locate a suitably polarised and EGFP expressing cell. The droplet was eased up to the cell membrane so that contact was made and left to incubate next to the cell for 10-15seconds. Faster incubation times tended to promote sufficient droplet -cell fusion and at greater incubation times droplets merged fully with the cell, preventing removal from the surface. Cell-droplet interactions were monitored using a combination of bright-field and fluorescence microscopy.

Figure 11 depicts an experiment where trypsonised BE human carcinoma cells expressing EGFP labelled CAAX proteins were pipetted onto a clean coverslip followed by the addition of lOμl of diluted droplet solution. Tether formation was observed following a 10 second incubation time with the BE cell plasma membrane. As a control the laser beam was fixed on the cell membrane for a period of 10-15secs and moved away at the same rate, where upon no tether was observed.

Tethers were extended at speeds of 0.5μm/sec at laser powers of 100-15OmW (forces = 100-20OpN). Below 10OmW (force = 100 pN), extraction rate exceeding 0.5μm/s often resulted in droplet/tether detachment. Typical lengths of tether observed were between 10-30μm (>cell diameter), consistent with previous observations reported for tethers pulled from cochlear outer hair cells, neuronal growth cones and red blood cells using latex beads. The efficiency of droplet-plasma membrane tether formation was over 80% with failure mainly due to too lengthy incubation times where the droplets were observed to irreversibly fuse with the target cell irrespective of the trapping power. A key observation from figure 11 is the uptake of fluorescently labelled material from the cell, not just to the tether but onto the droplet itself.

Trypsinized cells provide a relatively facile target for tether formation as the process of trypsinization leads to the digestion of the protein machinery on the plasma membrane responsible for inter-cellular adhesion or indeed adhesion to a container surface. There is therefore likely to be low resistance to fusion. If the droplets are to be used as a proteomic tool for sampling the plasma membrane it is beneficial to restrict any such modifications to the cell surface, particularly with a view to conducting time dependent studies. To further explore the potential of droplets as a tool for sampling the plasma membrane of single cell systems we therefore examined their mode of interaction with adherent non-trypsinized polarized BE cells grown on coverslips (figure 12). Again a success rate of over 80% for tether formation and a similar rate of fluorescent material transfer was observed using identical experimental protocols.

A key observation in both the trypsinized and un-trypsinized cell experiments is the uptake of EGFP CAAX labelled proteins from the inner leaflet of the plasma membrane of the cell to not only the membrane tether but also to the surface of the droplet itself. This observation confirms that the membrane tether does not consist solely of phospholipid material from the plasma membrane but also the proteins contained therein leading to the model shown in figure 12. It should also be noted that the fluorescence signal from the droplet does not reach a threshold value during the 20 second contact time meaning that longer incubation would result in even greater uptake of protein material.

Example 3 - Phase Behaviour Studies

In order to assess the fuginicity of DOPC/DOPE/hexadecane droplets, the DOPC/DOPE/xs water phase diagram was constructed between 0 mol % DOPC/ 100 mol %DOPE/xs water through to 100 mol % DOPC/ 0 mol %DOPE/xs in steps of 10 mol %. DOPC and DOPE were obtained from Avanti Polar Lipids Inc. (Alabaster, AL) with their purity stated to be > 97% pure. Both were stored at -20⁰C before being lyophilized under vacuum for 48 hours and weighed as a function of time to ensure that any excess water was removed. To make a given DOPC/DOPE sample, each lipid was freeze dried, weighed and then dissolved in chloroform forming clear stock solutions of known concentration. In order to generate homogeneous binary mixtures these solutions were then pipetted together in a glass vial to give the desired molar ratio and mixed thoroughly by vortexing. The chloroform was then evaporated off under nitrogen gas and the sample put back onto the lyophiliser over night. Lipid mixtures were then transferred to a 1.5mm glass capillary and water added (75wt% water). The capillary was then spun down in a centrifuge sealed and homogenised by means of four freeze-thaw mixing cycles. The sealed capillary was then placed in the sample holder of the X-ray beam line and weighed before and after to ensure no loss of water.

The phase behaviour of this binary system was measured using Small Angle X-Ray Scattering (SAXS). SAXS measurements were conducted by means of an house custom-built X-ray beamline. A Bede Microsource™ (Durham, UK) generator was used to produce the focused Cu K_n (1.542A) radiation after filtering out the Kp (1.3922A) with a nickel filter coupled with low divergence (2mrad) monolithic polycapillary optics (X-ray Optical Systems, Inc.,XOS-Albany, New York, USA). A Gemstar HS intensified CCD (Photonic Science Ltd, Battle, UK) was used to record the 2-D diffraction patterns. The detector-PC interface was established using a digital LVDS (RS-644) Picasso PCI-LS framegrabber card from ARVOO Imaging products (Natherlands). Sample capillaries (1.5mm diameter) were located in a copper sample holder with peltier (Melcor, NJ, USA) driven temperature control supported by a circulating water heat exchanger. This assembly was able to control the sample temperature to an accuracy of ± 0.1 °C Typical exposure times were 30seconds.

The diffraction patterns were analyzed and X-ray measurements were calibrated with silver behenate (dooi = 58.38 A).

The role of compartmentalization played by biological membranes requires that cellular systems maintain a balance of lipids in-vivo such that complex mixture of lipids in cells forms flat bilayer arrangements despite the fact that the same is not necessarily true of the individual lipids in isolation. When mixed with water for instance, type O lipids such as phosphatidylcholine (PC) lipids (e.g. 1,2-dioleoyl- phosphatidylcholines) tend to form flat bilayer arrangements, whereas the type II phosphatidylethanolamines (PE) (e.g. 1,2-dioleoyl-phosphatidylethanolamine) tend to form curved interfaces where the plane of the headgroup curves towards the water leading to the formation of mesophases such as the inverse hexagonal phase.

The propensity of type II lipids to form curved mesophases is reflected in part by the non-deformed unstressed curvature of the membrane which is commonly referred to as the spontaneous curvature of the membrane bilayer, Jς^B. The effective spontaneous curvature of DOPE has been reported as -1/3 nm ¹ in contrast to that of DOPC which lies between -(1/20) nm^"1 and -(1/8.7) nm^'1. By coating droplets with binary DOPC/DOPE coatings it is possible to fine-tune the propensity of these assemblies to undergo fusion with a target membrane.

In order to assess the fusogenicity of the droplets the phase behaviour of the DOPC/DOPE excess water system was measured. Compositions observed to form an inverse hexagonal phase at room temperature or above were employed as coatings for the droplets. In both the case of the inverse hexagonal and fluid lamellar phase a minimum of three peaks was used to assign the nature of the mesophase. The Hn phase gave rise to Bragg peaks in the ratio 1:V3:2:V7 and the lamellar phase in the ratio 1:2:3. Samples between 100 mol % DOPC/excess water and 55 mol % DOPE/ 45 mol % DOPC /excess water exclusively formed the fluid lamellar phase between O⁰C and 75⁰C. At 60 mol % DOPE / 40 mol % DOPC/excess water a transition from the fluid lamellar phase was observed at 75°C with the transition temperature dropping sharply to 45⁰C at 65 mol % DOPE / 35 mol % DOPC /excess water and 25°C at 70 mol % DOPE / 30 mol % DOPC/excess water. For both the 65 mol % DOPE / 35 mol % DOPC /excess water and 70 mol % DOPE / 30 mol % DOPC/excess water samples the inverse hexagonal phase was found to co-exist with the fluid lamellar phase up to 6O⁰C and 55⁰C respectively. At 85 mol % DOPE / 15 mol % DOPC /excess water the inverse hexagonal phase was observed to co-exist with the fluid lamellar phase between 5⁰C and 25⁰C and samples containing greater than 85 mol % DOPE were observed to form only the inverse hexagonal phase over the range of temperatures examined. In the light of these results droplets with coatings containing a minimum of 75 mol % DOPE were considered. Example 4 - Partial Solubilisation of a Cell Membrane

The following procedure was carried out to demonstrate extraction of a cell membrane protein by contacting a cell with a droplet having a surfactant containing coating.

BE cells expressing membrane located Ras-GFP were grown on polylysin-treated cover slides overnight, in DMEM supplemented with 10% Foetal Calf Serum, 100IU/ml Penicillin and 100 μg/ml Streptomycin. Medium was removed, cells were washed and covered with PBS.

In order to form a droplet emulsion, in a 15ml Falcon tube ImI PBS (cone), 0.5ml cyclohexane and 40 ul Triton-X 100 were mixed and shaken vigorously until the solution became milky, indicating formation of an emulsion containing droplets. The droplet solution was diluted 10x in PBS.

Under a microscope (Nikon TE2000-E, x64 NA= 1.2, water immersion objective) approximately a nl volume of the droplet suspension was dropped from a borosilicate glass microinjection needle (-10 μm tip diameter) onto the coverslide, in the proximity of the cells.

A droplet was trapped with 15OmW trapping laser power (IPG Photonics, Ytterbium fibre laser at wavelength=1070nm, diffraction limited focussed laser spot size =1 micron) and moved towards and into contact with the cell membrane of the GFP expressing cells, whilst observing the cells with a fluorescent microscope (FTTC filter cube set). Contact of the droplet with the cell membrane was maintained for 5 minutes after which the droplet was moved away from the cell. As shown in Figure 5, fluorescence was detected in the circled droplet 50 following this procedure, demonstrating that partial solubilisation of the membrane has occurred, with fluorescent membrane protein being transferred to the droplet.

Example 5 -Trapping of Droplets and Contacting Cells within a Microfluidic Format IxIO⁵ adherent BE cells were grown (as described for Example 1) for 12 hours on a cover slide (50x24mm), restricting the culture area to Ix lcm with adhesive chamber borders. The cover slide was maintained with cells facing up on the bottom of a tissue culture dish (diameter 10cm), covered with 15 ml of cell culture medium.

A PDMS chip was provided comprising a first delivery channel for a droplet emulsion and two additional delivery channels (second and third delivery channels) for effecting the generation of sheath flows by delivery of PBS. The three delivery channels converge into a microfluidic sheath flow channel. The chip additionally comprises a cell chamber and a droplet transport channel (or linking channel) linking one of the second or third delivery channels with the cell chamber. In order to bond the cover slide to a PDMS chip, the contact area has to be dry and clean. The cell free area of the cover slide was wiped and dried, taking care that cells stay covered with medium. The adhesive chamber borders were removed and the PDMS chip was bonded to the cover slip. A glass slide comprising silicon inlets and a silicon outlet was bonded to the other side of the PDMS chip and plastic tubing was connected to the silicon inlets and outlet.

A syringe was filled with PBS and connected to second and third delivery channels. A second syringe was filled with droplet-containing emulsion, prepared as previously described for Example 2, and connected to the central first delivery channel.

Flow of PBS was initiated for 1-3 minutes at 0.3μl/min until flow is established in the sheath flow channel of the microfluidic device. Flow of droplet emulsion is then initiated. Once stream focussing is established within the microfluidic channel, optical trapping of single or multiple droplets can be achieved and droplets can be directed towards the cell culture chamber, upstream from the PBS stream. This is demonstrated by Figure 6 which shows trapping and isolation of <1 micron droplets and separation of the droplets from the sheath flow. In Figure 6, circles show the position of the trapped droplets and arrow 60 represents the movement of optical traps. Trapping was sustained up to sheath flows of <0.8μl/m with a trapping power 0.045W per trap.

Example 6 -Trapping of Droplets and Contacting Cells within a Microfluidic Format IxIO⁵ adherent BE cells were grown on a cover slide (50x24mm), in the microfluidic chip for 12-24 hours within a culture chamber of 0.8cm in diameter. This was maintained with cells facing up on the bottom of a tissue culture dish (diameter 5cm), covered with enough (10-20μl) of cell culture media to totally submerse the cells. A similar amount of PBS/media can also be added to the emulsion droplet delivery chamber to aid the balance of pressures in the chip.

A PDMS chip was provided comprising a storage chamber for a droplet emulsion and a cell culture chamber. In order to bond the cover slide to a PDMS chip, the contact area has to be dry and clean. A glass slide comprising silicon inlets and a silicon outlet was bonded to the other side of the PDMS chip and plastic tubing was connected to the silicon inlets and outlet.

A syringe was filled with PBS/cell culture media and connected to the dividing flow channel. A second syringe was filled with droplet-containing emulsion, prepared as previously described for Example 5, and connected to the droplet storage chamber. A third syringe was filled and connected to the cell culture chamber.

Flow of PBS in the central channel was initiated for 5-10 minutes at l-10μl/min with the stop valve closed at the outlet until air bubbles are removed from the microfluidic junction and pushed out of the device. These air bubbles can then be sequentially removed completely from the peek tubing system via the 4-way valves. Once all air bubbles are removed the stop valve at the outlet can be opened and the flow of droplet emulsion and media can be initiated in the chambers. Once emulsion droplets drift to the microfluidic junction, optical trapping of single or multiple droplets can be achieved and droplets can be directed towards the cell culture chamber. This is demonstrated by Figure 9 which shows trapping and isolation of <3 micron droplet from emulsion droplet chamber. In Figure 9, square shows the position of the trapped droplet and subsequent movement of the optical trap from the emulsion droplet chamber across the microfluidic junction to the cell culture chamber is depicted by the arrows. Trapping was sustained up to flows of <lμl/min with a trapping power 0.090W per trap.

It should be understood that the invention is susceptible to various modifications and alternative forms. The invention is not to be limited to the particular forms disclosed, but should cover all modifications, equivalents and alternatives falling within the spirit of the disclosure.

Claims

1. A method for manipulating a cell comprising trapping and moving a droplet to bring it into contact with the cell and allowing interaction of the droplet with the cell, wherein the droplet comprises an interior and an exterior, wherein one of the interior and exterior is hydrophobic and the other is hydrophilic.

2. A method according to claim 1, further comprising trapping or immobilising the cell prior to and during contact of the droplet with the cell.

3. A method according to claim 2, wherein trapping or immobilisation is achieved by optical trapping, aspiration based methods (e.g. micropipette aspiration), dielectrophoresis or by adherence of a cell to a surface or to another cell.

4. A method according to any preceding claim, wherein the interior of the droplet comprises a fluid core and the exterior of the droplet comprises a coating.

5. A method according to claim 4, wherein the fluid core of the droplet comprises a water immiscible liquid.

6. A method according to claim 5, wherein the water immiscible liquid is an oil, preferably an alkane, for example hexane, heptane, hexadecane or cyclohexane.

7. A method according to claim 5 or 6, wherein the fluid core comprises a mixture of two or more water immiscible liquids.

8. A method according to claim 4, wherein the fluid core comprises an aqueous liquid, preferably an ionic liquid.

9. A method according to any one of claims 4 to 8, wherein the coating is formed from an amphiphile, for example a lipid derivative such as a phosphatidylethanolamine (e.g dioleoyl-phosphatidylethanolamine), a phosphatidylcholine (e.g. 1, 2-deheptanoyl-sn-Glycero-3-phosphocholine or 1, 2- dioleoyl-sn-glycero-3-phosphocholine), or a surfactant such as octyl-glucoside, sodium dodecyl sulphate (SDS), triton X-100, CHAPS or Zwittergent 3-12 or a mixture thereof.

10. A method according to claim 9, wherein the coating comprises a phosphatidylethanolamine and phosphatidylcholine mixture.

11. A method according to any one of claims 4 to 10, wherein the coating comprises a biocompatible molecule, for example PEG (polyethyleneglycol) or a PEG labelled lipid.

12. A method according to any preceding claim, wherein the maximum diameter of the droplet is no more than 50 microns.

13. A method according to any of claims 1 to 3 or 12, wherein the droplet is formed from an amphiphile and has a micelle-like structure, with the hydrophobic portion of the amphiphile forming a hydrophobic interior and the hydrophilic portion of the amphiphile forming a hydrophilic exterior.

14. A method according to any preceding claim wherein trapping and movement of the droplet are controlled by use of one or more of optical trapping, an aspiration based method (e.g. micropipette aspiration) or dielectrophoresis.

15. A method according to claim 14 wherein trapping and movement of the droplet is controlled by use of optical trapping.

16. A method according to any preceding claim, wherein the method comprises the step of providing an emulsion comprising a plurality of droplets, from which a droplet is trapped and moved into contact with a cell.

17. A method according to claim 16, wherein the emulsion is generated by mixing water (or an aqueous solution such as brine), a water immiscible liquid as defined in claim 5 or 6 and an amphiphile or mixture of amphipiles as defined in claim 9 or 10 and agitating the mixture such that the hydrophobic effect drives generation of an emulsion comprising amphiphile coated droplets.

18. A method according to any preceding claim, comprising at least one step carried out in a microfluidic format.

19. A method according to any preceding claim, wherein the droplet comprises a reagent incorporated therein, wherein the reagent is, for example, a small molecule, a drug molecule, a fluorescent molecule or a fluorescently labelled molecule, a biological molecule (such as a protein, an antibody, a plasmid, a cell organelle, RNA or DNA), an amphiphile or an inorganic reagent (including quantum dot moieties).

20. A method according to any preceding claim, wherein manipulation of a cell comprises physical deformation of a cell membrane by moving the droplet into contact with the surface of a cell, maintaining contact of the droplet with the cell surface for time sufficient to allow the droplet to fuse with the cell membrane and moving the droplet away from the membrane so as to deform the membrane.

21. A method according to any one of claims 1 to 19, wherein manipulation of a cell comprises partial solubilization of the plasma membrane of a cell by contacting the plasma membrane with a droplet, wherein the coating of the droplet comprises a surfactant.

22. A method according to any one of claims 1 to 19, wherein manipulation of a cell comprises delivery of a reagent to a specific location on or within a cell by contacting the cell with a droplet comprising a reagent and allowing delivery of the reagent to the cell.

23. A method according to any one of claims 1 to 19, wherein manipulation of a cell comprises removal of material from a cell by moving the droplet into contact with the cell, allowing material from the cell to enter the droplet or form a complex with the coating of the droplet and moving the droplet away from the cell, thereby removing material from the cell

24. A method according to claim 23, wherein (a) the droplet has a coating incorporating a protease which acts to cleave a cell surface protein;

(b) the droplet has a coating comprising a surfactant which partially solubilizes the cell membrane, leading to complex fromation with membrane one or more components; or

(c) the droplet is a fusagenic droplet which is moved to bring it into contact with the cell for sufficient time to allow transfer of material between the droplet and the cell.

25. A method according to claim 23 or 24, wherein after movement away from the cell the droplet is analysed downstream, using any one or more of a variety of analytical techniques including mass spectrometry, electrophoresis based methods, multi-dimensional fluorescence imaging (with respect to either one or all wavelength, lifetime and polarisation and in either two or three dimensions), labelled readout using microarray technologies or optical finger printing technologies.

26. A method for removal of material from a surface or from suspension within a liquid, comprising trapping and moving a droplet to bring it into contact with a surface bearing a material or a suspended material and allowing partitioning of the material into the droplet, wherein the droplet comprises an interior and an exterior, wherein one of the interior and exterior is hydrophobic and the other is hydrophilic.

27. A droplet for use in a method of cell manipulation, wherein the droplet has a maximum diameter of 50 microns and wherein the droplet comprises a fluid core and a coating and wherein the fluid core comprises a water-immiscible liquid and the coating comprises an amphiphile and wherein:

(a) the water immiscible liquid is an oil preferably an alkane, for example hexane, heptane, hexadecane or cyclohexane and the coating comprises an amphophilic lipid molecule or lipid derivative; or (b) the water immiscible liquid is heptane and the coating comprises an amphiphilic surfactant.

28. A droplet according to claim 27, wherein the coating comprises a lipid derivative such as a phosphatidylethanolamine (e.g dioleoyl- phosphatidylethanolamine) or a phosphatidylcholine (e.g. 1, 2-deheptanoyl-sn- Glycero-3-phosphocholine or DOPC) or a mixture thereof.

29. A droplet according to claim 28, wherein the water immiscible liquid is hexadecance and the coating comprises a mixture of DOPE and DOPC, wherein at least 75mol% of the mixture comprises DOPE.

30. A droplet according to any one of claims 27 to 29, wherein the coating comprises a biocompatible molecule, for example PEG (polyethyleneglycol) or a PEG labelled lipid.

31. A droplet according to any one of claims 27 to 30, wherein the maximum diameter of the droplet is in the range of 50nm to 5 microns.

32. A droplet according to any one of claims 27 to 31, wherein, the droplet comprises a reagent incorporated therein.

33. An emulsion comprising a plurality of droplets for use in a method for manipulation of a cell, wherein the droplets are as defined in any of claims 27 to 32, dispersed within an aqueous phase.

34. A microfluidic assembly comprising a first delivery channel for delivery of an emulsion comprising droplets as defined in any preceding claim, second and third delivery channels for delivery of a solvent, a microfluidic channel having a first end at which the first, second and third delivery channels converge and a second end provided with an outlet or a storage reservoir, a chamber and a linking channel which extends between one of the second or third delivery channels, upstream from the microfluidic channel, and the chamber.

35. A microfluidic assembly comprising a first channel extending between a first inlet connected to an emulsion storage chamber and second inlet, wherein the second inlet connects the first channel to a chamber, and a dividing flow channel extending between a third inlet and an outlet, wherein the dividing flow channel intersects with the first channel so as to form a microfluidic junction and wherein the first channel comprises a first portion extending between the first inlet and the microfluidic junction and a second portion extending between the microfluidic junction and the second inlet.

36. A microfluidic method for the isolation of a droplet as in any preceding claim, the method comprising providing a sheath flow comprising a plurality of droplets, trapping (preferably by optical trapping) one or more of the droplets and moving the one or droplets against the direction of sheath flow to an outlet positioned upstream of the sheath flow.

37. A method, droplet or microfluidic assembly substantially as described herein with reference to or as illustrated in any of the examples or figures of the accompanying drawings.