WO2015032900A1

WO2015032900A1 - A microfluidic device for cell culture observation and manipulation

Info

Publication number: WO2015032900A1
Application number: PCT/EP2014/068952
Authority: WO
Inventors: Devrim KILINC; Gil Lee; Walter Kolch; Stefano Rampini; Robert Franz SCHWAMBORN
Original assignee: University College Dublin, National University Of Ireland, Dublin
Priority date: 2013-09-05
Filing date: 2014-09-05
Publication date: 2015-03-12
Also published as: GB201315771D0

Abstract

A microfluidic device for cell culture observation comprises a chamber having sides that define a generally equilateral polygonal area configured for cell culture observation, and two or more microfluidic flow channels in fluid communication with the chamber, each microfluidic flow channel being arranged parallel to a side of the chamber. A transport barrier is disposed between each side and each microfluidic flow channel and configured to provide transport of solute from the microfluidic flow channel to the chamber along the side of the chamber.

Description

Title

A microfluidic device for cell culture observation and manipulation Field of the Invention

The invention relates to a microfluidic device for cell culture observation and manipulation. In particular, the invention relates to microfluidic culture platforms for studying cell behaviour when subjected to an active compound.

Background to the Invention

Concentration gradients are common in cell physiology and developmental biology. Extracellular gradients, for example, are necessary in directing cell migration or in axonal growth cone guidance; whereas intracellular gradients are necessary for a range of physiological activities from establishing polarity in cell division to maintaining the blood brain barrier.

Several methods have been employed for establishing concentration gradients in vitro, in order to simulate the physiological gradients experienced by the cells. These include the micropipette discharge method, and various chambers such as those named after Boyden, Zigmond, and Dunn. However, these classical methods do not provide stable concentration gradients that are highly desirable for biological experiments. Microfluidic cell culture devices solve most of the problems associated with these methods by providing precise geometric constraints for flowing solutions with known concentrations.

Flow-based microfluidic gradient generators rely on the diffusion of solutes through the boundaries of laminar flow streams that run parallel to each other. When a cascade of flow streams differing in starting concentrations are induced over a cell culture area, a concentration gradient forms that is orthogonal to the flow direction. By changing the pressure or flow rates of the inlet streams, one can manipulate the gradient steepness as well as the cell culture area that is exposed to gradients. The major disadvantage of flow-based gradient generators is that the cells are exposed to fluid shear stresses, which may affect their physiology and behaviour.

Diffusion-based microfluidic gradient generators rely on the passive diffusion of solutes from a source to a sink. Fick's law of diffusion states that a linear concentration gradient forms between a source and a sink, provided that their respective concentrations remain constant. This principle has been applied to a variety of microfluidic device designs tailored to experimental needs. These include point source devices, parallel chambers connected via microchannels or separated by hydrogel filling, a single chamber supplied by buried source channels, and devices designed for three-dimensional cell culture. Diffusion-based gradient devices do not expose cells to fluid shear stresses and hence provide a cell-benign system, suitable for cell biology research.

Classical biochemical methods are currently used in combination with simple, one-directional concentration gradient generation devices. Another approach is to establish a very large number of cell culture areas, i.e., micro-droplet arrays, and test combinations of molecules at varying concentrations. A synergistic/antagonistic study requires a high number of samples and is inherently limited in its power to resolve optimal concentration combination (say, for two drugs acting simultaneously). Therefore experiments using classical methods while exposing cells to a simple concentration gradient remain ineffective and inaccurate for testing a variety of interventions simultaneously.

Similarly, experimental methods relying on combinatorial application of molecules with variable concentrations do not have the capacity to impose a concentration gradient, which may be essential for the incitement of a morphological and behavioural response in the cells.

The US Patent Application 2009/0311737 (hereinafter referred to as the 'Locascio document'), discusses the use of a microfluidic disc which is capable of delivering multi- gradients of compounds simultaneously by diffusion into a chamber which holds a population of cells. The cells can be monitored for long periods of time to assess the cells behaviour to the compounds. Both bacterial and mammalian cells can be used with this disc. The system described in the Locascio patent is based on point contacts between the channels and the chamber. The resulting gradients are demonstrated as diffusion from a point source, which results in non-linear concentration gradients emanating from all directions and angles. Therefore, it is impossible to have zones of uniform size that have defined concentrations, which is a requirement for reliable cell-based experiments.

Further microfluidic culture systems such as that described in US2011217771A1 focus on the 3D cell culture and a single concentration gradient across a polymeric matrix. International Publication WO 2012/033439A1 focuses on sustained cell culture exposed to a single concentration gradient. However, neither system are effective nor accurate for testing a variety of interventions simultaneously. There are other publications such as EP 2127748, EP 1508373, WO 2009/101850 and WO 2010/080978 which demonstrate linear or non-linear gradients. However, these are specifically for a single compound in a single direction.

There is a growing need for combinatorial assay systems in cell biology and pharmacology. It is an object of the present invention to overcome at least one of the above-mentioned problems.

Summary of the Invention

The invention is focused on increasing the speed and decreasing the cost of drug discovery through the development of micro-reactors and micro-devices that can produce well-defined linear chemical gradients of one or more compounds. The device comprises a chamber defined by at least four sides that define a generally equilateral polygonal area that is suitable for receiving cells. Well-defined linear gradients of one or more compounds are generated in the chamber by the transport of the compound into the chamber along at least two adjacent edges of the chamber of the device (Figs. 2 and 12). A device capable of creating multiple linear concentration gradients simultaneously facilitates experiments testing the synergistic/antagonistic interaction of molecules, such as drugs or inhibitors, on cells. It also facilitates the rapid identification of the dose response of the cells to potential drugs and inhibitors.

In a first aspect, the invention provides a microfluidic device for cell culture observation comprising:

a cell chamber having four or more sides that defines a generally equilateral polygonal area configured for cell culture observation;

two or more microfluidic flow channels in fluid communication with the cell chamber, each microfluidic flow channel being arranged parallel to a side of the cell chamber; and a transport barrier disposed between each side and each microfluidic flow channel and configured to provide transport of solute from the microfluidic flow channel to the cell chamber along the side of the cell chamber. Typically the cell chamber is an even-sided, convex, equilateral regular polygon.

Typically, the transport barrier comprises a barrier having a plurality of microchannels or pores. Each microchannel is suitably lnm-200 microns long, 5nm-15 microns wide, and 100nm-5 microns high. Typically, each microchannel is 150-200 microns long, 5-15 microns wide, and 1-5 microns high. Preferably, each microchannel is 190-210 microns long, 8-12 microns wide, and 2-4 microns high. In one embodiment, the transport barrier comprises a single pore traversing the width the barrier.

Preferably, the device is configured to provide transport of solute into the cell chamber by means of passive diffusion. Alternatively, the device may be configured to provide transport of solute into the cell chamber by alternative means, for example by means of capillary action or convection.

In one embodiment, the at least two or more adjacent microfluidic flow channels are in fluid communication with each other.

Suitably, the at least two adjacent microfluidic flow channels comprise a different inlet port and the same outlet port. In one embodiment, the at least two or more adjacent microfluidic flow channels are not in fluid communication with each other. Suitably, the at least two or more adjacent microfluidic flow channels comprise a solute storage vessel in fluid communication with the cell chamber, with the microfluidic flow channel delivering solute from the vessel to the cell chamber. The storage vessel is configured to act as the inlet port, while the outlet port is redundant (see Figure 13(B)). The term "solute storage vessel" should be understood to mean a hollow space in the device that is accessible directly from outside of the device and is in direct fluid communication with the transport barrier via the microfluidic flow channel. The solute storage vessel may be sealed from above (like the bubble trap as depicted in Figure 13C), to induce flow of solute in to and out of vessel. In one embodiment, the device further comprises a bubble trap positioned between the inlet port and the flow channel. In the specification, the bubble trap is defined as an enclosed chamber with an inlet channel placed on its one side and an outlet channel placed on its opposite side. Gas bubbles entering the bubble trap are pushed upwards due to the buoyancy force and therefore cannot be present in the outlet channel, hence becomes "trapped" in this chamber.

Preferably, the cell chamber has an area of less than 0.0001m², more preferably less than 0.00006 m². In one embodiment, the cell chamber has a width of less than 10, 9, 8, 7, 6, 5 or 4 mm.

Suitably, the device further includes at least one reservoir for fluid, generally cell culture fluid, and at least one inlet port adapted to provide fluid communication between the reservoir and the cell chamber. Generally, the at least one inlet port provides fluid communication between the reservoir and a corner of the cell chamber. Suitably, the device comprises at least two reservoirs and at least two inlet ports, wherein the each inlet port provides fluid communication between one reservoir and one corner of the cell chamber. Suitably, device comprises one outlet port for each inlet port, or one outlet port for every two inlet ports, or one outlet port for all inlet ports. Where the solute storage vessel is used in place of the inlet port, there is no outlet port.

In one embodiment, the device is coated with a silica monolayer. The monolayer is preferably applied through employing the sol-gel method. Suitably, the silica monolayer is selected from tetraethyl orthosilicate, methyltriethoxysilane, or trimethoxyboroxine. Ideally, the silica monolayer is tetraethyl orthosilicate.

In one embodiment, the shape of the even- sided regular polygonal area of the cell chamber is selected from a square, a hexagon, an octagon, a decagon, a dodecagon, a tetradecagon, or any similar shape suitable for providing two or more linear gradients of a molecule. Suitably, the device has four sides and is square- shaped.

The invention also provides a microfluidic system for monitoring cell culture comprising the microfluidic device according to the invention. The system may additionally include pumping means for providing a flow of solute through at least two of the microfluidic flow channels. In one embodiment, the pumping system is configured to provide a flow of fluid to all of the microfluidic flow channels. Typically, the pumping system is configured for adjustment of the pressure of the solute flowing through the or each microfluidic flow channel. Suitably, the pumping system is configured for independent control of the pressure of the flow of fluid to the or each flow channel. Since pressure and flow rate are linearly related to each other for a given channel geometry, and since the flow channel geometries in the device of the invention are substantially identical, the term pressure and flow rate can be used interchangeably. Typically, the pumping means is configured for pumping a first fluid through first adjacent microfluidic channels simultaneously, and at the same pressure or flow rate, such that a linear concentration gradient forms throughout the cell chamber in a first direction.

Suitably, the pumping means is configured for pumping a second fluid through second adjacent flow channels simultaneously, and at the same pressure or flow rate, such that a second linear concentration gradient forms throughout the cell chamber in a direction different to the first direction.

Thus, for example, the first fluid may contain a first molecule, and the second fluid may contain a second molecule, such that a linear concentration gradient of each molecule is set up in the cell chamber in which the linear concentration gradients have different directions.

The first adjacent microfluidic flow channels and second adjacent flow channels may partially overlap (whereby one flow channel from each pair is arranged along the same side of the cell chamber - See Fig. IB - a, a+b, b). In this arrangement, the first linear concentration gradient is orthogonal to the second linear concentration gradient.

Alternatively, the first adjacent microfluidic flow channels and second adjacent flow channels do not overlap at all. In this arrangement, the first linear concentration gradient is in a direction that is opposite to the second linear concentration gradient.

It will be apparent that in this manner up to four different concentration gradients may be set up in the square cell chamber, all having different directions. In another aspect, the invention provides a method for observing cell culture in the presence of a one-directional linear concentration gradient of at least one test molecule, which method comprises providing a microfluidic device of the invention in which the cell culture is provided within the cell chamber, providing a fluid comprising the test molecule to two adjacent flow channels such that the test molecule is transported into the cell chamber along two adjacent sides of the cell chamber thereby generating a linear concentration gradient of the test molecule in the cell chamber along a first direction, and observing the response of the cells within the cell chamber to the linear concentration gradient of the test molecule. The transport of the test molecule may be driven by passive diffusion between the source and the sink, by osmotic pressure, or by convective mass transfer.

The invention also provides a method for observing cell culture in the presence of a one- directional linear concentration gradient of at least two test molecules, which method comprises providing a microfluidic device of the invention in which the cell culture is provided within the cell chamber, providing a first fluid comprising a first test molecule to first adjacent flow channels such that the first test molecule is transported into the cell chamber along first adjacent sides of the cell chamber thereby generating a linear concentration gradient of the first test molecule in the cell chamber along a first direction, providing a second fluid comprising a second test molecule to second adjacent flow channels such that the second test molecule is transported into the cell chamber along second adjacent sides of the cell chamber thereby generating a linear concentration gradient of the second test molecule in the cell chamber along a second direction, and observing the response of the cells within the cell chamber to the linear concentration gradients of the test molecules. The invention also provides a method for observing cell culture in the presence of a one- directional linear concentration gradient of at least three test molecule, which method comprises providing a microfluidic device of the invention in which the cell culture is provided within the cell chamber, providing a first fluid comprising a first test molecule to first adjacent flow channels such that the first test molecule is transported into the cell chamber along first adjacent sides of the chamber thereby generating a linear concentration gradient of the first test molecule in the cell chamber along a first direction, providing a second fluid comprising a second test molecule to second adjacent flow channels such that the second test molecule is transported into the cell chamber along second adjacent sides of the cell chamber thereby generating a linear concentration gradient of the second test molecule in the cell chamber along a second direction, providing a third fluid comprising a third test molecule to third adjacent flow channels such that the third test molecule is transported into the cell chamber along third adjacent sides of the cell chamber thereby generating a linear concentration gradient of the third test molecule in the cell chamber along a third direction, and observing the response of the cells within the cell chamber to the linear concentration gradients of the test molecules.

The invention also provides a method for observing cell culture in the presence of a one- directional linear concentration gradient of four test molecules, which method comprises providing a microfluidic device of the invention in which the cell culture is provided within the cell chamber, providing a first fluid comprising a first test molecule to first adjacent flow channels such that the first test molecule is transported into the cell chamber along first adjacent sides of the cell chamber thereby generating a linear concentration gradient of the first test molecule in the cell chamber along a first direction, providing a second fluid comprising a second test molecule to second adjacent flow channels such that the second test molecule is transported into the cell chamber along second adjacent sides of the cell chamber thereby generating a linear concentration gradient of the second test molecule in the chamber along a second direction, providing a third fluid comprising a third test molecule to third adjacent flow channels such that the third test molecule is transported into the cell chamber along third adjacent sides of the chamber thereby generating a linear concentration gradient of the third test molecule in the cell chamber along a third direction, providing a fourth fluid comprising a fourth test molecule to fourth adjacent flow channels such that the fourth test molecule is transported into the cell chamber along fourth adjacent sides of the chamber thereby generating a linear concentration gradient of the fourth test molecule in the cell chamber along a fourth direction, and observing the response of the cells within the cell chamber to the linear concentration gradients of the test molecules.

The invention also provides a method for observing cell culture in the presence of a one- directional linear concentration gradient of six test molecules, which method comprises providing a microfluidic device of the invention in which the cell culture is provided within the cell chamber, providing a first fluid comprising a first test molecule to first three adjacent flow channels such that the first test molecule is transported into the cell chamber along first three adjacent sides of the cell chamber thereby generating a linear concentration gradient of the first test molecule in the chamber along a first direction, providing a second fluid comprising a second test molecule to second three adjacent flow channels such that the second test molecule is transported into the cell chamber along second three adjacent sides of the cell chamber thereby generating a linear concentration gradient of the second test molecule in the cell chamber along a second direction, providing a third fluid comprising a third test molecule to third three adjacent flow channels such that the third test molecule is transported into the cell chamber along third three adjacent sides of the cell chamber thereby generating a linear concentration gradient of the third test molecule in the chamber along a third direction, providing a fourth fluid comprising a fourth test molecule to fourth three adjacent flow channels such that the fourth test molecule is transported into the cell chamber along fourth three adjacent sides of the cell chamber thereby generating a linear concentration gradient of the fourth test molecule in the chamber along a fourth direction, providing a fifth fluid comprising a fifth test molecule to fifth three adjacent flow channel such that the fifth test molecule is transported into the cell chamber along fifth three adjacent sides of the cell chamber thereby generating a linear concentration gradient of the fifth test molecule in the cell chamber along a fifth direction, providing a sixth fluid comprising a sixth test molecule to sixth three adjacent flow channels such that the sixth test molecule is transported into the cell chamber along sixth three adjacent sides of the cell chamber thereby generating a linear concentration gradient of the sixth test molecule in the cell chamber along a sixth direction and observing the response of the cells within the cell chamber to the linear concentration gradients of the test molecules.

The shape of the cell chamber of the device can vary depending on the number of test molecules being used. However, the cell chamber of the device can be any shape having a sufficient even-numbered sides so as to permit the testing of the desired number of molecules. For example, a square-shaped cell chamber is sufficient to test from 1 to 4 molecules, a hexagon- shaped cell chamber is sufficient to test from 1 to 6 molecules while an octagon is sufficient to test from 1 to 8 molecules.

Suitably, the first, second, third, fourth, fifth and sixth directions are different directions. The different directions may be orthogonal to each other, or opposite to each other, when the cell chamber is square-shaped. In a further embodiment, the different directions may be at a 60 degree angle to each other when the cell chamber is hexagonal (using 3 adjacent sides, up to 6 directions), a 45 degree angle when the cell chamber is octagonal (using 4 adjacent sides, up to 8 directions), a 36 degree angle when the cell chamber is decagonal (using 5 adjacent sides, up to 10 directions), a 30 degree angle when the cell chamber is dodecagonal (using 6 adjacent sides, up to 12 directions), or a 25.7 degree angle when the cell chamber is tetradecagonal (using 7 adjacent sides, up to 14 directions). In all instances, the concentration gradients are linear irrespective of the shape of the chamber. The use of a hexagon is illustrated in Figure 12 (o, ci, cic₂, cic₂c₃, cic₂c₃c₄, c₂c₃c₄, c₃c₄, c₄).

The response of the cells may be determined in any manner known to those skilled in the art, including visual observation (i.e. migration, morphology, cell cycle analysis), or monitoring the phenotype of the cells using a diagnostic reagent, for example a fluorophore-labelled cytoskeletal marker, a cytoplasmic dye or a cell death marker. Immunocytochemistry may be applied to live, alcohol-, or aldehyde-fixed cells to visualize and/or quantify proteins of interest through the use of specific antibodies.

In one embodiment, the method of the invention is a method for determining the response of cells, typically tumour cells, to a combination of molecules, at least one of which, and ideally all of which, are anti-cancer drugs, for example 2, 3 or 4 or more anti-cancer drugs. This method may be employed to design a cocktail of drugs that is most suited for treatment of a certain tumour. Thus, it is envisaged that the tumour cells are derived from a patient biopsy. In this manner, tumour cells from the patient may be rapidly exposed to different combinations, concentrations, or both combinations and concentrations, of drugs, and the response of the tumour cells to the different combinations may be determined. In an alternative embodiment, the tumour cells may be cancer cell lines, for example colon cancer cell line HCT-116, breast cancer cell lines MCF7, or prostate cancer cell line PC3. The device will also enable the comparative screening of cell lines with different genetic mutation profiles for differential, mutation dependent sensitivities against compounds and combinations of compounds.

The method of the invention may also be employed to determine the nutritional status or requirements of a sample of cells, in which the test molecules are cell substrates, for example carbon, nitrogen or sulphur sources.

The method of the invention may also be employed to determine the toxicity of one or more test molecules to a sample of cells. In another embodiment, the method of the invention may be used to expose three- dimensional cell cultures to concentration gradients of one or more molecules. The three- dimensional culture, i.e., isolated cells embedded in a biocompatible polymer matrix, may be formed in the cell culture chamber.

In yet another embodiment, the method of the invention may be used to expose cells to concentration gradients of one or more molecules in an open well format, where the elastomeric material over the cell culture chamber is removed to deem it directly accessible, rather than through the reservoirs.

In a further embodiment, the method of the invention may be multiplexed to match the standard 6, 12, 24, 48, or 96 well microtitre plate formats for high content imaging and analysis applications. In a preferred embodiment, a multitude of cell culture chambers may be aligned with microtitre plate well positions. In an alternative embodiment the inlet flow channels may be connected to feed a multitude of cell culture chambers with the same liquid carrying one or more molecules.

This invention is based on the transport of molecules from parallel flows from four or more microfluidic flow channels that run parallel to the edges of a modified polygon-shaped cell (cell) culture chamber. The flow channels and the cell culture chamber are typically interconnected via micro-channels to act as a transport barrier. When fluid flow is induced through the flow channels simultaneously and at the same pressure or flow rate, mass transport will be limited to molecular diffusion. If a molecule is constantly supplied to the two adjacent sides of the square shape and not to the remaining sides, a linear concentration gradient of this molecule will form throughout the cell culture chamber in one direction. By applying this principle to multiple directions, four or more linear concentration gradients can be formed in a single device. Cells exposed to the gradients can be (i) observed for extended periods (>8h) for migration/morphology/cell cycle analysis; (ii) induced with live fluorescent reporters for monitoring cell signalling; (iii) fixed and immunostained with antibodies of interest. This device is suitable for long-term live cell microscopy and classical immunocytochemistry.

The invention can be integrated with on-chip pressure and concentration sensors situated at the inlet flow manifolds. This will help regulate the formed linear gradients independent of the efficiency of the fluidic connections and inequalities in the device and access port dimensions. The invention can be integrated with on-chip reagent reservoirs and passive pumping methods. The invention can be tested for the efficiency of the fluidic connections and inequalities with the use of tracers, such as different colour dyes and the like.

The method of the invention may also be employed to assess calcium transport or use by cells, the pH of the cellular environment of the cells, the number of cells in a given area etc.

Definitions

In the specification, the term "cell" should be understood to mean any cell of eukaryotic or prokaryotic origin, including cells of animal, plant, bacterial, or fungal origin. The term should also be understood to encompass unmodified and genetically modified cells, healthy cells and cells representative of a disease state, and cells that are stressed or unstressed. In a preferred embodiment of the invention, the cells are mammalian cells, preferably cancer- derived cells. In one embodiment, the cells are tumour cells from a cancer biopsy. In this specification, the term "cancer" should be understood to mean a cancer selected from the group comprising: esophagogastric cancer; fibrosarcoma; myxosarcoma; liposarcoma; chondrosarcoma; osteogenic sarcoma; chordoma; angiosarcoma; endotheliosarcoma; lymphangiosarcoma; lymphangioendotheliosarcoma; synovioma; mesothelioma; Ewing's tumour; leiomyosarcoma; rhabdomyosarcoma; colon carcinoma; pancreatic cancer; breast cancer; ovarian cancer; prostate cancer; squamous cell carcinoma; basal cell carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinomas; cystadenocarcinoma; medullary carcinoma; bronchogenic carcinoma; renal cell carcinoma; hepatoma; bile duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilms' tumour; cervical cancer; uterine cancer; testicular tumour; lung carcinoma; small cell lung carcinoma; bladder carcinoma; epithelial carcinoma; glioma; astrocytoma; medulloblastoma; craniopharyngioma; ependymoma; pinealoma; hemangioblastoma; acoustic neuroma; oligodendroglioma; meningioma; melanoma; retinoblastoma; and leukemias. In the specification, the term "microfhiidic flow channel" should be understood to mean a space or combination of spaces that forms between a flat substrate and an elastomeric pad bonded to it, whose height is less than 100 μιη. For example, a channel, a canal, a groove, and the like.

In the specification, the term "molecule" or "test molecule" should be understood to mean any compound or molecule, including but not limited to chemical compounds or conjugates, biological molecules such as proteins, peptides, sugars, lipids, antibodies, antibody fragments, hormones, growth factors, nucleic acids (including DNA and RNA molecules), low molecular weight compounds, including miRNA and siRNA molecules, and conjugates of any of these molecules.

In the specification, the term "transport" should be understood to mean the convective and diffusive mass transport of a substance subject to conservation laws of physics. In the specification, the term "limited transport" should be understood to mean the mass transport that is hindered by imposed geometrical or physical constraints.

In the specification, the terms (i) "osmotic pressure", (ii) "passive diffusion" and (iii) "convective mass transfer" should be understood to mean, in the context of movement of the test molecule between the source and the sink, (i) osmotic pressure is the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane, (ii) the molecular transport of one substance relative to another (also known as mass diffusion, concentration diffusion, or ordinary diffusion), (iii) the transport of a substance by the bulk transport of the fluid which the substance is in.

In the specification, the term "bubble trap" should be understood to mean an enclosed chamber with an inlet channel placed on its one side and an outlet channel placed on its opposite side. Gas bubbles entering the bubble trap are pushed upwards due to the buoyancy force and therefore cannot be present in the outlet channel, hence becomes "trapped" in this chamber.

Brief Description of the Drawings The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:-

Figure 1 illustrates A. Top view of the microfluidic device showing the cell chamber (dark grey), flow channels (light grey) and microchannels connecting the two (black). B. Flow configurations to obtain orthogonal gradients. Molecule a flows through the left flow channels and forms a horizontal gradient. Molecule b flows through the top flow channels and forms a vertical gradient. When molecule a, molecule b, their mixture, and control medium flows according to the depicted configuration, an orthogonal gradient forms. C. 25 regions that were used to measure the concentration distribution within the cell chamber.

Figure 2 illustrates the finite element analysis results in terms of concentration distributions as heat maps at indicated time points for low (A) and high (B) molecular weight molecules.

Figure 3 illustrates the finite element analysis results in terms of the evolution of the concentration gradient over time for low (A) and high (B) molecular weight molecules. The cell culture chamber is situated between 400 μιη and 2000 μιη marks in this analysis.

Figure 4 illustrates the establishment of the linear concentration gradient in the cell chamber of the microfluidic device. A. 25-region maps of rhodamine concentration (C*, normalized with bulk flow concentration) at different time points. B. Column averages were plotted against jc-position in the chamber for indicated time points. C. Concentration profiles at each time point were fit with straight lines (C* = dCldx · (x - LIT) + C*so). Time profiles of dC^*/dx and C*so (dC* value at x = LIT) show the dynamic formation of the rhodamine gradient.

Figure 5 illustrates live cell caspase activation experiments in A431 cells treated with 0.1 nM EGF. A. The experimental paradigm showing orthogonal gradients of U0126 and BIBX in the x- and _ -directions, respectively. Cells were treated with uniform concentrations of EGF. B. 25-region maps showing the percentage of cells with activated caspase 3/7 at indicated time points during simultaneous U0126 (left-to-right, decreasing) and BIBX (top- to-bottom, decreasing) treatment. C. Column and row averages of the apoptosis rate (percentage of cell with activated caspase) were fit with straight lines to calculate the x- and _ -gradients, respectively, of the change in the apoptosis rate. Data presented is the average of two independent experiments. Error bars represent the standard error.

Figure 6 Live cell caspase activation experiments in A431 cells treated with 10 nM EGF. A. The experimental paradigm showing orthogonal gradients of U0126 and BIBX in the x- and _y-directions, respectively. Cells were treated with uniform concentrations of EGF. Broken brown line indicates the regions considered as the bottom half of the chamber. B. 25- region maps showing the percentage of cells with activated caspase 3/7 at indicated time points during simultaneous U0126 (left-to-right, decreasing) and BIBX (top-to-bottom, decreasing) treatment. C. Column and row averages of the apoptosis rate (percentage of cell with activated caspase) were fit with straight lines to calculate the x- and _ -gradients, respectively, of the change in the apoptosis rate. In addition, jc-gradient of the change in the apoptosis rate for bottom half of the chamber is given. Data presented is the average of three independent experiments. Error bars represent the standard error.

Figure 7 illustrates live cell motility experiments. A. The experimental paradigm showing orthogonal gradients of EGF and BIBX in the x- and _ -directions, respectively. Quadrants are marked with brown broken lines. Cell images are taken at 10 h time point in boxed areas C6 (left) and E6 (right). Phase contrast overlay with green fluorescence. Scale bars = 100 μιη. B. 25-region maps of the average magnitude of cell velocity vector for given time periods during simultaneous EGF (left-to-right, decreasing) and BIBX (top-to-bottom, decreasing) treatment. C. Average vector magnitudes calculated for each quadrant for given time periods. D. Average vector velocities normalized with that obtained in Q₄. E. Column and row averages were fit with straight lines to calculate the x- and _ -gradients, respectively, of the change in the vector magnitude. Data presented is the average of three independent experiments. Error bars represent the standard error.

Figure 8 illustrates A. Cell death rate for varying TRAIL concentrations with 1 μg/ml CHX. B. Cell death rate for varying CHX concentrations with 3-10 ng/ml TRAIL. C. Comparison of TRAIL-induced cell death with (black) and without (white) 1 μg/ml CHX at various TRAIL concentrations.

Figure 9 illustrates a time course of PI staining in HeLa cells in the orthogonal gradient device. Cells death is evident around 2 h in cells exposed to TRAIL (top-to-bottom, increasing, bulk concentration = 3 μg/ml) and CHX (left-to-right, decreasing, bulk concentration = 3 μg/ml) in the regions close to flow channels with TRAIL. By 10 h, low TRAIL concentration regions of the chamber also exhibit Pi-positive cells.

Figure 10 illustrates Pi-positive HeLa cells in different quadrants of the chamber at 1 h (white), 3 h (gray), and 4 h (black) time points.

Figure 11 illustrates the difference between non-linear and linear gradients. The thick line symbolizes equilibrium state (for example, after 8hr). With a non-linear gradient, the concentration change per position change (dC/dx) varies greatly depending on the distance from the source. With the linear gradient, dC/dx is constant, hence, uniform distances for a certain range of concentration (or uniform areas for certain ranges of concentrations in 2D) are attainable.

Figure 12 illustrates that when the lines connecting the edges of a polygon cross in a single point, they become symmetry axes of the polygon and linear gradients form perpendicular to these lines.

Figure 13 illustrates (A) a device of Figure 1 further comprising bubble traps (BT), four inlets (Ii to I₄) and a common outlet (O); (B) a device of Figure 1 where the microfluidic flow channels comprise a solute storage vessel in place of the inlets; and (C) a cross-sectional view of a bubble trap.

Detailed Description of the Drawings

Materials and Methods

Microfluidic device design and fabrication

The microfluidic circuit (Fig. 1A) consists of an extended square- shaped cell chamber (6 mm x 6 mm; 100 μιη high) and four microfluidic flow channels (200 μιη wide; 100 μιη high) running parallel to the edges of the cell chamber. Flow channels are connected to the cell chamber with parallel microchannels (200 μιη long; 10 μιη wide; 3 μιη high; 20 μιη spacing). Two step photolithography was used to fabricate the master pattern on a 4 inch silicon wafer (University Wafer, Boston, MA): First, a 3 μιη layer of SU-8 2002 (MicroChem, Newton, MA) was spincoated using a spin-coater (Laurell Technologies, North Wales, PA) and exposed to UV light (365 nm; 14.8 mW) for 30 s through a photomask containing microchannel geometry (Micro Lithography Services, Essex, UK). Following development of the pattern in EC developer (Chestech, Bristol, UK), a 100 μιη layer of SU-8 2010 (MicroChem) was spin-coated. Using a mask aligner (Suss MicroTec, Garching, Germany) the second layer of photoresist was exposed to UV light for 60 s through a photomask containing the rest of the geometry (Micro Lithography). Following development of the second layer in EC developer, the master pattern was treated with chlorotrimethylsilane (Sigma Aldrich, St. Louis, MO). The first generation of polydimethysiloxane (PDMS, Sylgard 184; Dow Corning, Midland, MI) pads were fabricated via replica molding from the master pattern. Polyglass (Artificina, Puteaux, France) replicates were fabricated using the first generation pads. Replicates were used to fabricate subsequent generations of PDMS pads. 1.5 mm access holes were punched to the opposite ends of the cell compartment using biopsy punches (Ted Pella, Redding, CA). Likewise, 0.75 mm access holes were punched to the inlet and outlets of flow channels. The pad was then permanently bonded to a 50x24 mm glass coverslip via oxygen plasma (Plasma Etch, Carson City, NV). The chamber and the flow channels were wetted and the device was sterilized by exposing it to UV light for 20 min. The device can be stored in a humid environment for several days.

A solute storage vessel can be used in place of the inlets (see Figure 13B). In this instance, the solute storage vessel comprises a shallow dish to act as a fixed-volume source or sink for the molecules of interest, with the microfluidic flow channel spreading out from an edge of the vessel like a wide-mouthed channel. The microfluidic flow channel provides the fluid communication between the vessel and the cell chamber, thereby allowing one or more concentration gradients to form between opposite solute storage vessels containing different concentrations of solute. Suitably, one of these concentrations is zero.

Finite element simulations

Diffusion of molecules in the microfluidic device was simulated using Comsol engineering platform. A three-dimensional finite elements model was created that includes the cell (culture) chamber, diffusion barriers, and flow channels. However, for reasons of simplicity, only those portions of the flow channels that are parallel to the cell culture chamber are modelled. Flow of solution carrying a diffusing species was simulated by dictating a pressure difference between the inlets and outlets of each flow channel segment. The initial value of the concentration of this species was set to 1 whereas the initial value was set to 0 for the rest of the model including the inlet flow solution that did not contain this species. Two different diffusion coefficients (2.3xl0^-11 and 2.6xl0^~10) for the transported species in order to match literature values given for the diffusion of FITC and 70kDa Dextran in PBS. The simulation was run for 8 h and the concentration distribution in the cell culture chamber was displayed as heat maps (Fig. 2A and 2B). The concentration along the x-coordinate was also measured during post-processing at different time points to demonstrate the formation of the gradient in a dynamic way (Fig. 3A and 3B).

Bubble Traps and Fluidic Assembly

Bubble trap devices containing four reservoirs each were placed in the inlet flow line to eliminate bubbles entering the microfluidic devices during experiments. The bubble trap devices were fabricated by first preparing rectangular PDMS pads of 4 mm thickness and punching out four through holes with a 4 mm diameter biopsy punch. Then, the pads were placed on a semi-cured (12 min at 70°C) PDMS layer of 1 mm thickness and the PDMS layer was fully cured (2h at 70°C). Inlet and outlet holes were punched using 0.5 mm diameter biopsy punches with an angle of ca. 45°, where the outlet hole joined the reservoir at a lower position than the inlet hole. Bubble traps were bonded to the same glass coverslips on which the microfluidic devices were bonded, (see Figure 13A).

Inlet flows were induced by a syringe pump (KR Analytical, Cheshire, UK) with four identical syringe units. Syringes (Hamilton, Bonaduz, Switzerland) were connected to the inlet holes of the bubble traps with luer connectors and PEEK tubing (Upchurch Scientific, Oak Harbor, WA) whose outer diameter is slightly larger than the inlet hole diameter. Outlet flows from the bubble traps were connected to inlet holes of the microfluidic device. Outlet flows from the microfluidic device were diverted to a third PDMS pad where the two flows were merged, such that the outlet pressures were equal, and released to a waste collector. This pad was bonded on a glass slide coated with pluronic F-127 (Sigma) to avoid droplet formation.

To block the medium reservoirs at either end of the cell chamber during experiments, 1/16" O.D. tubing was connected with a plug (Upchurch). In order to generate a linear concentration gradient of a molecular species across the cell chamber, bulk concentration of this species was delivered to two adjacent flow channels. This species was absent from the remaining two flow channels. In order to induce orthogonal gradients of two different species, the same arrangement is juxtaposed with 90° angle (Figure IB).

Characterization of concentration distribution

The distribution of the concentration within the microfluidic device was measured as a function of time using fluorescent tracers with molecular weights matching small inhibitory drugs: fluorescein isothiocyanate (FITC; Sigma) and rhodamine-B (Sigma). The microfluidic device was placed on the stage of an inverted microscope (Axioscope Zl; Zeiss, Cambridge, UK) equipped with an EMCCD camera (Hamamatsu, Herts, UK). The microscope was controlled by Axiovision imaging software (Zeiss) and maintained in the housing of a temperature controller (Life Imaging Services, Basel, Switzerland). First, the relationship between the concentration and signal intensity was established by filling the cell chamber with constant concentrations of tracers. The top surface of the microfludic device was covered with light absorbent vinyl film (VWR, Dublin, Ireland) to eliminate light reflecting from this surface. To measure the gradients, the four inlets were supplied with 125 μΜ FITC in PBS, 125 μΜ FITC and 125 μΜ rhodamine in PBS, 125 μΜ rhodamine in PBS, and PBS alone, such that both FITC and rhodamine were supplied through the two adjacent flow channels. 25 regions mapping the cell chamber were registered in the imaging software for automated acquisition (Fig. 1C). Images were acquired every 4 min for 14 h.

Cell culture and treatments

MDA-MB-231 breast carcinoma cells expressing GFP in their nuclei and A431 epidermoid carcinoma cells were cultured in growth medium: DMEM containing 3.97 mM L-glutamine and 25 mM glucose (Gibco, Carlsbad, CA), 10% fetal bovine serum (FBS, Gibco), and an antibiotic mixture of 100 U/ml penicillin and 100 μg/ml streptomycin (Pen/Strep). Cells were passaged every 3-4 days. Two days before the experiment, cells were harvested by trypsinization and suspended in growth medium at a density of 7.5xl0⁴ and 5xl0⁵ cells/ml for MDA motility and A431 apoptosis experiments, respectively. 16 μΐ of cell suspension was then introduced into the cell chamber via one reservoir and sucked from the other side, such that the chamber was filled uniformly. For efficient cell attachment, the devices were incubated for 30 min before the access wells were topped with medium. Cells were incubated at 37 °C by placing the micro fluidic device in a plastic Petri dish containing 1 ml 0.1% ethylendiaminetetraacetic acid EDTA in dH₂0 to minimize evaporation. One day after seeding cells were starved by replacing the full medium with DMEM containing Pen/Strep. 4-6 h prior to the experiment, the medium was replaced again with Leibovitz's LI 5 medium (Gibco) containing Pen/Strep.

A431 cells were treated with uniform concentrations of human EGF (Roche, Clarecastle, Ireland) which was added to the cell chamber, as well as included in all four flow media. A431 cells were subjected to orthogonal concentration gradients of BIBX 1382 dihydrochloride (Tocris Bioscience, Bristol, UK) and U0126 (Promega, Madison, WI), selective inhibitors of EGF receptor tyrosine kinase and MEK, respectively. Bulk concentrations of U0126, BIBX, and EGF were 50 μΜ, 25 μΜ, and 0.1 or 10 nM, respectively. MDA cells were subjected to orthogonal concentration gradients of human EGF and BIBX 1382. Bulk concentrations of BIBX and EGF were 25 μΜ and 50 nM, respectively. Detection of Caspase Activation

30 min prior to the experiment, A431 cells were labeled with CellEvent caspase-3/7 green detection reagent (Invitrogen) by replacing the medium in the microfluidic device with 10 μΜ of dye solution in L15 medium. This dye is a fluoreogenic substrate for activated caspase 3/7, which is considered a crucial event in the induction of apoptosis. The cell chamber was imaged at 25 positions every 20 min for 12 h, using brightfield and green epifluorescence for caspase detection. Caspase-positive cells were identified by a sudden increase in the fluorescent signal intensity from one time point to the next one. Percentages of caspase- positive cells were calculated for all time points at all positions. The average of at least three independent experiments were reported for each condition.

Cell motility analysis

25 registered positions in the cell chamber were imaged every 20 min for 16 h while MDA cells were exposed to orthogonal EGF/BIBX gradients. Movements of individual cells were analyzed by using the MTrackJ plugin of the ImageJ software (NIH, Bethesda, MD) by tracking their nuclei using a filter set suitable for GFP. Cell motility was expressed in terms of velocity vector magnitude, which is equal to the net displacement of the nucleus during the indicated time period.

Cell polarization is determined by the relative alignment of the Golgi apparatus and the nucleus after a 4h-long exposure to the orthogonal EGF/BIBX gradient. Cells are immunostained with GM130 antibody and subsequently with a FITC-conjugated secondary antibody, and their nuclei are labeled with Hoechst 33342. 25 registered positions in the cell chamber are imaged and the orientation of individual cells determined using the ImageJ software (NIH, Bethesda, MD).

Flow cytometry

For flow cytometry experiments, HeLa cells were harvested 3 to 4 h after TRAIL and/or CHX treatment via trypsinization. Detached cells were washed twice with ice-cold PBS and incubated in PBS with 2 μg/ml PI for 30 min on ice. An Accuri 6 flow cytometer was used to collect data and to conduct subsequent analyses. The experiment was repeated in triplicate.

Apoptosis assay Either 2 μg/ml propidium iodide (PI; Sigma) or 1 μΜ TO-PRO®-3 (Molecular Probes, Eugene, OR) were added to the L15 medium to detect cell death. Experiments were performed on an inverted Nikon Eclipse Ti-E microscope equipped with XYZ Prior stage and Andor Revolution spinning disc.

For FRET analysis, CFP (445 nm excitation; 475nm emission) and YFP (515 nm excitation; 526 nm emission) filter sets are used. PI and TO-PRO®-3 fluorescence are measured through 560 nm/624 nm and 594 nm / 624 nm filter sets, respectively. Either 4x or lOx objectives are used, with 2x2 and 4x4 binning, respectively. EM gain was set at 250. The MC protocol of the Andor IQ software is used to perform an XY scan of the chip area. Assembled images are analysed using ImageJ. For analysis, the chip area is segmented into 25 regions as described. Cell death nuclear dye is deemed positive when intensity values are at least three times higher than background intensity. Caspase 3 FRET probe cleavage is analysed by comparing at FRET/YFP and CFP/YFP intensity ratios.

Results and Discussion

Finite element simulations predict the formation of linear concentration gradients

Finite element simulations were conducted to calculate the concentration distribution with the cell culture chamber as a function of time for two different solute diffusion coefficients representing low and high molecular weight tracers. A smaller cell culture chamber was modelled to decrease computational requirements; yet, analysis results demonstrate the formation of the linear gradient within the chamber as a function of time and tracer molecular weight. Figure 2 shows the concentration distribution in the cell culture chamber at select time points for each tracer. Figure 3 shows concentration vs. distance plots for select time points for each tracer at the horizontal centreline of the cell culture chamber. It takes approximately 45 min and 5 h for low and high molecular weight molecules to reach a stable gradient, respectively. In summary, the finite element simulations suggest a linear concentration gradient to form as a result of the preferred flow configuration, i.e., tracer flow in two adjacent flow channels and tracer- free flow in the remaining flow channels.

Concentration gradients form in a time- and molecular weight-dependent manner

The concentration of the tracer molecule, C, within the cell chamber is expressed in terms of percentage of the bulk concentration flowing in the flow channels. Fig. 4A shows the concentration distribution of rhodamine at selected time points. The concentration profile established in the early hours after the start of the induced flow and remains stable through the duration of the experiment (Fig. 4B). These profiles are fit with lines such that the data is represented in terms of two variables: the gradient dC/dx, expressed in - mm^"1, and the mid-chamber concentration C50, expressed in %. The concentration at a given position x and time t can then be calculated from C (t,x) = C50 (t) + dC/dx (t) · (x - LIT), where L/2 is half width of the cell chamber. Figure 4C shows the time profiles of dC/dx and C50, for rhodamine tracer.

EGF induces apoptosis in A431 cells which synergistically increases with co-treatment with MEK inhibitors

EGF at high concentrations (10 nM) induces apoptosis in A431 cells, an epidermoid carcinoma cell line that over-expresses EGF receptors. Preliminary off-chip experiments suggested that MEK inhibition increases EGF-induced apoptosis in A431 cells in a synergistic fashion. A431 cells were exposed to uniform concentrations of EGF (0.1 nM or 10 nM) by adding EGF in the cell chamber as well as providing it through the four flow channels. Simultaneously, orthogonal linear gradients of BIBX and U0126, specific inhibitors of EGF receptor and MEK, respectively, were formed by supplying the flow channels with these molecules (Fig. 5A, 6A). Caspase 3/7 activation, an early marker of apoptotic induction, was monitored for 8 h at different locations in the cell chamber. Apoptosis rate was expressed in terms of percentage of cells with activated caspase 3/7. Figure 5B shows the caspase activation maps for select time points where the cells appear to undergo apoptosis in the left-hand side of the device, i.e. , where the U0126 concentration is high. When the data is analyzed in terms of column averages and row averages, a gradient in the jc-direction can be seen (Fig. 5C). Apoptosis rate gradient in the jc-direction, expressed as the increase in the percentage of apoptotic cells per mm, was constant during the course of experiment, suggesting the successful formation of the U0126 gradient. In the y-direction where the BIBX gradient was formed, no gradient can be detected if the row averages are considered. However, when the right-hand side of the cell chamber is considered, a subtle difference in the apoptosis rate can be observed: Cells that received low U0126 and high BIBX did not show any caspase activation, whereas cells that received low U0126 and low BIBX exhibit some level of caspase activation towards the end of the imaging period.

The rate of apoptosis increased considerably when the cells were exposed to EGF at high concentrations. The caspase activation maps in Fig. 6B show a clear delay in apoptosis in cells receiving high concentrations of BIBX (top of the chamber). Accordingly, the y- gradient of the apoptosis rate has a negative value throughout the course of the experiment (Fig. 6C). This shows that EGF-induced apoptosis can be blocked by inhibiting EGF receptors in a dose-dependent manner. In the jc-direction, no obvious difference can be detected between the cells receiving high U0126 and low U0126. However, when the analysis is constrained to the bottom half of the cell chamber (indicated with brown dashed line in Fig. 6A), a subtle jc-gradient (more apoptosis in cells receiving high U0126) becomes evident at later time points, suggesting a small additive interaction between EGF stimulation and MEK inhibition.

MDA cells respond to EGF stimulation and EGF receptor inhibition in dose-dependent fashion

To demonstrate the capacity of the orthogonal gradient device for monitoring cell motility over extended periods, a well-studied experimental paradigm, cell chemotaxis towards EGF was studied. MDA cells with fmorescently-tagged nuclei were treated with orthogonal gradients of EGF and BIBX (Fig. 7A). Cell motility was expressed in terms of velocity vector magnitude, the net distance that the cell nucleus moved during a period of time divided by that period of time. Heat maps of the average cell motility were shown for four consecutive 4 h-long intervals (Fig. 7B). Due to the inherent variations in motility in these non-synchronized cells, the data was analyzed by dividing the cell chamber into four quadrants, indicated by brown broken lines in Fig. 7A. Cells in quadrant 1 (Qi) were exposed to high levels of EGF and high levels of BIBX, cells in quadrant 2 (Q₂) were exposed to high levels of EGF and low levels of BIBX, cells in quadrant 3 (Q₃) were exposed to low levels of EGF and high levels of BIBX, and cells in quadrant 4 (Q₄) were exposed to low levels of EGF and low levels of BIBX. The changes in the motility was expressed in absolute velocity vector magnitudes (Fig. 7C) and after normalizing the velocity vector magnitudes in each quadrant with that of Q₄ (Fig. 7D), the quadrant that received low EGF and low BIBX. The relatively low motility in Qi is evident within the first 4 h time period. The motility in Qi further decreases between 8 h and 12 h time periods. In contrast, cells in Q₃ exhibit a delayed decrease in their motility. Cell motility in Q₃ was similar to Q₄ levels during the first 4 h time period, but gradually decreased from then on and was similar to Qi levels at the end of the 16 h experiment. This suggests that inhibition of EGF receptor by BIBX blocks the motility of MDA cells. The dynamics of this process depends on the availability of EGF to the cells, where cells exposed to high EGF slow down faster; however, eventually the cells receiving high and low EGF exhibit similar motilities. These observations are further confirmed when the column and row averages of vector magnitude values were fitted with lines to calculate the gradients (Fig. 7E). Here, the motility gradient in the _ -direction, i.e., in the same direction with the BIBX concentration gradient, shows an increasing trend, whereas the motility gradient in the x- direction is very close to zero.

The effect of EGF alone can be seen when Q2 and Q4 are compared since these quadrants receive low levels of BIBX but varying levels of EGF. There is a subtle yet gradual decrease in the cell motility with higher levels of EGF. However, cells in these quadrants exhibited striking differences in the cell morphology at late time points (Fig. 7A). Cells exposed to high EGF exhibited more mesenchymal shapes, whereas cells exposed to low EGF were rounder. The lack of cell chemotaxis towards high EGF regions may be explained in the light of detailed studies showing that the EGF gradient detection in MDA-MB-231 cells require nonlinear gradient profiles. Since the microfluidic system presented here generates linear gradient profiles only, cell chemotaxis was not observed. Nevertheless, these results suggest that the orthogonal gradient device provides a suitable platform for conducting experiments based on cell morphology and motility data.

CHX has a synergistic effect on TRAIL-induced cell death

A set of flow cytometry experiments were performed to verify the synergistic cell death- induction by TRAIL and CHX and to determine optimal concentration ranges that result in the most significant difference in the cellular response. HeLa cells were first treated for 3-4 h with varying TRAIL concentration at constant CHX concentration (1 μg/ml) (Figure 8A). The cells were then treated with either 3 or 10 ng/ml TRAIL, the concentration range that gave the largest difference in cell death, at varying CHX concentrations (Figure 8B). Finally, the synergistic effect of CHX on cell death was assessed at different TRAIL concentrations (Figure 8C). These results suggest that CHX has a small but distinct synergistic effect on TRAIL-induced cell death. Target concentration ranges for TRAIL and CHX to be used in the linear gradient generator were identified based on the flow cytometry data.

Linear gradients of TRAIL and CHX demonstrate synergistic induction of cell death To achieve a TRAIL concentration gradient in the 10 - 100 ng/ml range (equivalent to 1 - 10 ng/ml of high-potency TRAIL, not shown) across the cell chamber at 2 h time point, the bulk concentration of TRAIL was set to 3 μg/ml. Similarly, to achieve a CHX concentration gradient in the 0.006 - 0.7 μg/ml range at this time point, the bulk concentration of CHX was set to 3 μg/ml. Periodic images of the cell chamber shows that PI staining emerges in high TRAIL areas of the chamber at around 3 h and progresses towards low TRAIL areas (Figure 9). To compensate the low number of cells, the cell death analysis was conducted by dividing the chamber into 4 non- overlapping quadrants as indicated in Figure 9 (white triangles). Cell death rates in Sham, CHX only, TRAIL only and TRAIL+CHX quadrants at 1, 3, and 4 h time points are given in Figure 10. At 3 h time point, there is a clear difference between TRAIL only and TRAIL+CHX quadrants (46.7% vs. 73.6%) indicating a synergistic effect between CHX and TRAIL. This effect can also be observed at 4 h time point, but to a lesser extent (75.2% vs. 89.5%). Within this treatment model, small concentration changes of 1: 10 lead to big changes in cell response. CHX is much smaller than TRAIL (0.3 kDa vs. 19 kDa) and thus diffuses faster over the chip area. Yet as its inhibition is reversible, it does not accumulate. Based on these experimental parameters, easily detectable differences in cell response within the same device can be observed.

Advantages of the invention are that uniform concentration of an agent(s) along the entire edge of the connections of the chamber result in linear gradients parallel to the axis can be attained. The chamber can be divided into distinct zones, whose concentration values are well defined. Quantitative cell biology experiments are possible to implement due to having a uniform zone with defined concentrations of one or more agents.

A further advantage of the invention is that the device has the capacity to create a large number of combinations of molecules and their concentrations and can also create a suitable test bed to study morphology, signalling, and behaviour of cells exposed to molecular gradients.

Another major advantage of using this system for drug discovery-related research is that the use of the microfluidic devices and the reduction in the number of test samples reduces the total amount of reagents consumed, thereby improving the cost- and time-effectiveness of the drug discovery process.

In the specification the terms "comprise, comprises, comprised and comprising" or any variation thereof and the terms "include, includes, included and including" or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa.

The invention is not limited to the embodiments hereinbefore described but may be varied in both construction and detail.

Claims

1. A microfluidic device for cell culture observation comprising:

two or more microfluidic flow channels in fluid communication with the cell chamber, each microfluidic flow channel being arranged parallel to a side of the cell chamber; and

a transport barrier disposed between each side and each microfluidic flow channel and configured to provide transport of solute from the microfluidic flow channel to the cell chamber along the side of the cell chamber.

2. A microfluidic device as claimed in Claim 1 in which the transport barrier comprises a barrier having a plurality of microchannels or pores.

3. A microfluidic device as claimed in Claim 1 or Claim 2 in which at least two adjacent microfluidic channels are in fluid communication with each other.

4. A device according to any one of Claims 1 to 3, wherein at least two adjacent microfluidic flow channels further comprise a different inlet port and the same outlet port.

5. A device according to any one of the preceding claims, further comprising a bubble trap positioned between the inlet port and the flow channel.

6. A device according to any one of Claims 1 to 2, wherein the at least two adjacent microfluidic flow channels are not in fluid communication with each other and further comprise a solute storage vessel in fluid communication with the cell chamber.

7. A device according to any one of the preceding claims, in which the cell chamber has an area of less than 0.0001m².

8. A device as claimed in any preceding Claim, and further including at least one reservoir for fluid and at least one inlet port adapted to provide fluid communication between the reservoir and the cell chamber.

9. A device according to any one of the preceding claims, in which the device is coated with a silica monolayer.

10. A device according to Claim 10, wherein the silica monolayer is selected from tetraethyl orthosilicate, methyltriethoxysilane, or trimethoxyboroxine.

11. A device as claimed in any preceding Claim, wherein the equilateral polygonal area is selected from the group comprising a square, a hexagon, an octagon, a decagon, a dodecagon, a tetradecagon.

12. A microfluidic system for monitoring cell culture comprising the microfluidic device according to any one of the preceding Claims.

13. A microfluidic system according to Claim 12, including means for providing the same fluid to first adjacent microfluidic flow channels simultaneously, and at the same pressure, such that a linear concentration gradient forms throughout the cell chamber in a first direction.

14. A microfluidic system according to Claim 13, including means for providing a different fluid to second adjacent microfluidic flow channels simultaneously, and at the same pressure, such that a second linear concentration gradient forms throughout the cell chamber in a direction different to the first direction.

15. A microfluidic system as claimed in Claim 13 and 14 in which the first adjacent microfluidic flow channels and second adjacent microfluidic flow channels do not overlap, and wherein the first linear concentration gradient is parallel to the second linear concentration gradient.

16. A microfluidic system as Claimed in Claim 12 and 13 in which the first adjacent microfluidic flow channels and second adjacent microfluidic flow channels partially overlap, and wherein the first linear concentration gradient makes a certain angle with the second linear concentration gradient.

17. A method for observing cell culture in the presence of a one-directional linear concentration gradient of at least one test molecule, which method comprises providing a microfluidic device as claimed in any of Claims 1 to 11 in which the cell culture is provided within the cell chamber, and providing a fluid comprising the test molecule to two or more adjacent flow channels such that the test molecule is transported into the cell chamber along two or more adjacent sides of the cell chamber thereby generating a linear concentration gradient of the test molecule in the chamber along a first direction, and observing the response of the cells within the cell chamber to the linear concentration gradient of the test molecule.

18. A method for observing cell culture in the presence of a one-directional linear concentration gradient of two or more test molecules, which method comprises providing a microfluidic device as claimed in any of Claims 1 to 11 in which the cell culture is provided within the cell chamber, providing a first fluid comprising a first test molecule to first adjacent flow channels such that the first test molecule is transported into the cell chamber along first adjacent sides of the chamber thereby generating a linear concentration gradient of the first test molecule in the cell chamber along a first direction, providing a second fluid comprising a second test molecule to second adjacent flow channels such that the second test molecule is transported into the cell chamber along second adjacent sides of the cell chamber thereby generating a linear concentration gradient of the second test molecule in the chamber along a second direction, and observing the response of the cells within the cell chamber to the linear concentration gradients of the test molecules.

19. A method for observing cell culture in the presence of a one-directional concentration gradient of three test molecule, which method comprises providing a microfluidic device as claimed in any of Claims 1 to 11 in which the cell culture is provided within the cell chamber, providing a first fluid comprising a first test molecule to first adjacent flow channels such that the first test molecule is transported into the cell chamber along first adjacent sides of the cell chamber thereby generating a linear concentration gradient of the first test molecule in the chamber along a first direction, providing a second fluid comprising a second test molecule to second adjacent flow channels such that the second test molecule is transported into the cell chamber along second adjacent sides of the chamber thereby generating a linear concentration gradient of the second test molecule in the cell chamber along a second direction, providing a third fluid comprising a third test molecule to third adjacent flow channels such that the third test molecule is transported into the cell chamber along third adjacent sides of the chamber thereby generating a linear concentration gradient of the third test molecule in the cell chamber along a third direction, and observing the response of the cells within the cell chamber to the linear concentration gradients of the test molecules.

A method according to any one of Claims 17, 18 or 19 in which first, second and third directions are different directions.

A method according to any of Claims 17 to 20 for determining the response of cells to a plurality of anti-cancer drugs, in which the cells are tumour cells, and in which the test molecules are different anti-cancer drugs, and wherein the reaction of the tumour cells to different combinations of the drugs is monitored.