This invention relates to a device and method for quantifying the linear cell migration rate of individual cells, in particular mammalian cells, over a substrate from one location to another.

Background

The ability to move, specifically to translocate, is a fundamental property of cells in both prokaryotic and eukaryotic organisms. Cell migration is the outcome of a concerted series of intracellular events, generally initiated by signals from the extracellular environment. These signals may include chemicals, capable of activating cell surface receptors or can be environmental cues such as pH, temperature or oxygen concentration.

Cell migration has a fundamental role in the development and maintenance of eukaryotic organisms, with aberrant cell migration being implicated in the progression of pathogenic states such as cancer. Cells possess structure that is governed by the cytoskeleton; an internal network of various structural proteins. An actively migrating mammalian cell exhibits polarity and orientates with a distinct leading edge and a corresponding trailing edge. Substrate attached cells possess localised points of close contact between the cell and underlying substrate, known as focal adhesions. These consist of a distinct cluster of cell surface receptors (typically the integrin family of receptors) which are involved in tethering the cell to the surface. Focal adhesions not only provide mechanical attachment but also have a biochemical signalling function by the transduction of signals from the external environment; via the cell surface receptors to the internal components of the cell such as the cytoskeleton. This cytoskeleton provides 'shape' to the cell, and since it is subject to dynamic regulation and reorganisation it can exert control over the cells shape and polarity. A migrating cell uses its cytoskeleton to extend protrusions in the direction of migration, and to actively withdraw the trailing edge. Simultaneously, via the regulated control of polymerisation and turnover of actin molecules the cell increases focal adhesions at the leading edge and decreases them at the trailing edge. The result is translocation over the substrate.

Several techniques are available to quantitatively measure cell migration. Boyden Chamber Assay

This utilises a thin membrane (typically polycarbonate or polyethylene), perforated with many pores of a defined diameter to form a cell permeable barrier between an upper and lower chamber. Cells in culture media are placed in the top compartment. The bottom compartment contains a chemoattractant (such as conditioned media). A gradient of chemoattractant molecules exists between the two chambers. Upon incubation at physiological conditions the cells migrate through the pores to the source of chemoattractant and colonise the membrane underside. At the end of the incubation non-migrated cells are removed from the upper surface of the membrane using a moistened cotton bud tip. The cells on the membrane underside are immobilised through chemical fixation before being stained for analysis. Quantification of migrated cells, located on the membrane underside is usually achieved through manual cell counts using a microscope. The researcher can test the effect of treatment agents on the cells by placing them in solution either above or below the membrane depending on the particular agent being tested. However, use of the Boyden Chamber is time-consuming and laborious. WO 01/32827 utilises the Boyden Chamber design principles, but the membrane is replaced by a silicon wafer. The silicon wafer includes passages through which cells undergoing chemotaxis can move. In the device of WO01/32827, gravity causes cells to move towards the openings of the passages. A hopper shaped portion at the entrance to the passages guides such cells to the opening aperture of the passage in the membrane. The cells undergoing chemotaxis move into the passages in the wafer and cells which pass through the exit aperture of the passage are detected. Wound Healing or Cell Outgrowth Assays

These are an alternative to trans-membrane migration assays. In these systems, cells are allowed to form a uniform, confluent monolayer in a defined area, then, either a barrier is removed or the monolayer is mechanically wounded. Both approaches permit cell migration and proliferation into a new, clear area. The progress of the cells can be monitored microscopically as they migrate and proliferate into the cell free zone. Treatment agents can be added in solution to determine the effect on cell migration. Quantitative analysis is achieved either by measurement of the population diameter or by cell counts at selected areas over time.

Although this assay is relatively simple to perform it does not distinguish between the processes of cell migration and cell proliferation.

The Dunn Chamber Chemotaxis Assay

This consists of a glass slide with two concentric wells located in the centre, separated by a slightly raised 'bridge'. The outermost well is filled with culture medium containing chemoattractant, and the inner well filled with culture medium only. Cells are cultured at low density on a glass coverslip which is then inverted over the two wells and sealed around the edges. In this closed system a radially directed linear chemoattractant gradient is formed running across the 'bridge' area of the chamber. The assay system is incubated for up to 24 hours on a microscope heated stage. Images of the cells in the chemoattractant gradient region are recorded at defined intervals over the duration of the experiment. The cell images are then subjected to computer image analysis, the outcome being a 'vector' plot of each individual cells movement. These plots are overlaid to produce an overall diagram of cell migration in response to the chemoattractant. The assay procedure is complex and requires investment in a time-lapse video microscope with a heated stage. The assay is not suited to large scale experiments or indeed parallel experiments since one microscope set-up is required for each individual experiment.

Phagokinetic Track Assay

This assay relies on the ability of migrating cells to phagocytose particles coated onto a migration substrate, the track area being proportional to the magnitude of cell migration. The particles are non-toxic, typically composed of gold or fluorescent latex beads. Quantification is achieved by computer image analysis of the cell tracks to calculate the cell area and hence distance migrated. The set-up and analysis of the assay can be extremely time-consuming.

A number of devices described in the literature utilise microchannels in which the cells can migrate, see for example EP1340810, US2003/0022362 and US 2003/0017582. However, it is not considered these devices control the location of cells in the vicinity of the microchannel entrance such that the cells can be observed undergoing chemotaxis in the microchannel.

There is a need for an alternative assay to measure cell migration. Summary of Invention

According to a first aspect of the invention, there is provided a cell migration measurement device comprising a substrate member with:

at least one access port in a surface of the substrate adapted to receive a fluid sample;

at least one exit port adapted to output fluid sample from the device;

at least one internal surface of the device defining at least one microchannel in the substrate extending from and connected to the access port and the exit port of the device for flow of the fluid sample through the microchannel from the access port to the exit port,

wherein the internal surface which defines the at least one microchannel comprises a supply channel in fluid connection with a cell receiving portion, a constriction portion and a cell migration measuring portion, the cell receiving portion being adapted to substantially receive and present a predetermined number of cells in suspension to the constriction portion and the constriction portion being interposed between the cell receiving portion and cell migration measuring portion and the constriction portion being of a dimension that a cell in fluid suspension provided to the at least one microchannel via the access port cannot pass the constriction portion into the cell migration measuring portion, but a cell with a morphology providing for attachment to the internal surface and therefore with decreased cross- sectional area can pass the constriction portion into the cell migration measuring portion, wherein the cell receiving portion comprises multiple hopper shaped cell receiving portions provided in fluid connection with the supply channel, wherein each of the cell receiving portions provide cells provided in the fluid sample to a constriction portion which is in fluid connection with a cell measuring microchannel. The hopper shaped cell receiving portion has a wide mouth portion adjacent to the supply channel and narrows to end in the constriction portion. The hopper shaped cell receiving portion can be generally funnel shaped. The substantially horizontal alignment of the hopper relative to the measuring microchannel utilises hydrodynamic flow through the cell receiving portion. The hydrodynamic flow can generate a two-dimensional or planar distribution of cells resting on the cell receiving portion floor.

The present invention allows a defined proportion of the cells provided to the device to be provided in the cell receiving portion of each measuring microchannel. Typically around 20 to 30 cells will be located in the cell receiving portions of the device and these cells can then move into the measuring microchannel following substrate attachment of the cells and the ensuing cell migration. This is advantageous as it allows an array of similar microchannels to be considered a replicate of its neighbour and thus be directly comparable. Advantageously, the device of the present invention can allow a user to provide an aliquot of cells to the access port and without further intervention the device can allocate a defined quota of cells to multiple cell receiving portions. The cells are reproducibly aligned with the measuring microchannel at the start of an assay. Only once the biological process of cell attachment has begun may the cells traverse the constricted entrance of the microchannel to enter the measurement portion.

Such a device enables the measurement and examination of cell migration (the migratory rate of cells). It may also be used to determine the affect of reagents or exogenous compounds on cell migration. A further modification of the device allows for the determination of the affect of substrate immobilised compounds on the cell migration of cells provided to the device as a fluid sample.

In embodiments the cell receiving portion can be a hopper shaped portion of the microchannel which is provided in fluid connection with the supply channel wherein the supply channel provides a hydrodynamic flow such that it causes cells to accumulate at the constriction portion in the cell receiving portion prior to cell attachment to substrate and ensuing migration into the measuring channels. The constriction portion provides a passage from the cell receiving portion of the microchannel to the measuring portion of the microchannel, the passage having a cross sectional area that is smaller than the width of a substrate-detached cell in the cell population present in the cell receiving portion. In embodiments the width can be the mean cross-sectional diameter of a substrate detached cell. The cell receiving portion provides and presents a small cluster of cells to the constriction portion to ensure that cells move into the constriction portion and then into the measuring microchannel and minimises the chance of a cell moving back towards the access port. Without the cell receiving portion, the inventors have found that a single cell only may locate at the constriction portion and such a cell(s) may not enter the measuring microchannel.

In embodiments, the hydrodynamic flow that is responsible for cell loading in the cell receiving portion of the microchannel, for example hoppers of the microchannel array, can be generated by the differential height in fluid levels between the access port and the exit port of device, or between a loading reservoir and exit reservoir. Suitably, at the time of supply of substrate-detached cells in fluid suspension to the device (device loading) the difference in fluid head height may lie within the quotient range of 1.25 to 1.6. The quotient value relates the initial set reduction in fluid head height at the exit or exit reservoir relative to the fluid head height at the access, for example of the loading reservoir. This quotient is considered suitable to minimise shear stress to the cells and the risk that hydrodynamic flow will push cells past the constriction portion i.e. it ensures that only cells undergoing chemotaxis can move past the suitable sized constriction portion. The supply channel can be in fluid connection to multiple cell receiving portions, constriction portions and cell measuring microchannels. The cell receiving portions, constriction portions and cell measuring microchannels can be arranged in parallel, wherein for example a single supply channel is in fluid connection with 2, 3, 4, 5, 6, 7, 8, 9 or 10 cell receiving (shaped hoppers or funnels) and constriction portions leading to measuring microchannels. The advantage of providing cell receiving portions and constriction portions in a multiple parallel arrangement is that they functionally provide a loading system that can guarantee a similar level of cell loading in all of the measuring microchannels. This in turn results in a similar timing for each cell to move from the constriction portion and into the measuring microchannel and so ensures that each measuring microchannel acts as a close replicate of its neighbour.

In the present description, length is considered to be the dimension of the microchannel between the access port and exit port. As will be appreciated, depending on the shape of the microchannel, this length of the microchannel need not be a straight linear measument, but can be a length of a curved microchannel for example in embodiments the supply channel may be curved to allow preferred arrangements of the device to be accommodated on the substrate. In the present disclosure, width and height are orthogonal to length. Typically, width can be considered to be in an XY plane of the device as it would be provided in use and height as a height in Z when the device is in use. The length of the measuring microchannel may suitably be in the range 1000 to 2500 micrometers (microns or μηι). This allows the migration of the fastest anchorage dependent cell types within the desired assay time periods.

In preferred embodiments the measuring microchannel of the device is at least 20 micrometers in width between the walls formed by the internal surfaces which form the microchannel, as this minimises the likelihood of cells becoming highly elongated in form and migrating in full contact with both walls of the microchannel which makes observation and quantification difficult. A 20 micrometer wide microchannel allows a cell to migrate in a preferred mode - which is single wall attachment. A 20 micrometer wide microchannel also minimises cells crossing back and forth from one wall to another which may potentially slow the migration rates. Increasing the width of the microchannel is considered to reduce any shear stress on a cell in the microchannel but reduces the density of parallel microchannels which can be provided across the width of the substrate member of the device.

In a preferred embodiment the measuring microchannel can have a height of at least 20 micrometers. Although not wishing to be bound by theory, this height is considered to minimise the risk that any cell clumps (two or more cells attached together) may stick between a microchannel 'floor' and 'ceiling' as formed by the internal surfaces of the device forming the microchannel and block the device. In embodiments, the measuring microchannel can have a height of 25 micrometers and a width of 20 micrometers, more preferably 25 micrometers in width.

Suitably, a width and height combination of a 1:1 ratio may be utilised.

In a preferred embodiment the cross-sectional shape of a microchannel can be a rectangle or square with a 90° angle between a floor and wall of a microchannel. A 90° angle maximises the contact area of a cell in the microchannel between the floor and wall. Cells appear to preferentially migrate 'locked' into this 90° angle. In embodiments, an angle of less than 90° between the floor and wall of the microchannel may be provided. Alternatively, the wall can meet the floor as a smooth curve. A rectangular cross-section can be provided by photolithography of the substrate member. In embodiments, the constriction portion is sized to form a passage that has a cross- section less than the width or diameter of a cell or the mean width of a cell to be measured, for example a monolayer-derived cell, a primary cell or a cell from continuous culture. In particular, a cell(s) to be measured can be from a population of transformed cells i.e. tumour derived cells. Although each cell line possesses its own morphology, when released from a monolayer using enzymatic or detergent treatment the cells assume a rounded, generally spherical shape, and each occupy a similar volume.

Due to the limited plasticity of the fluid-suspended cells, the vertical height of the microchannel at the constriction portion may be 20 micrometers as a minimum height.

In embodiments, the constriction portion can form a passage for cells to move through wherein the passage is of about 3 to 8 micrometers in width, suitably 5 to 8 micrometers in width and about 20 micrometers in height. The constriction portion can extend for a portion of the microchannel of about 12.5 micrometers in length or more. For smaller cell types such as leukocytes, a constriction portion forming a passage of 3 micrometers in width should be used. In embodiments the length of the constriction portion can be at least 12.5 micrometers with a width of passage through which cells are to move of between 5 to 8 micrometers and a height of 20 micrometers. Whilst the length of the constriction portion can be increased in length to a maximum of 50 micrometers; there appears to be little advantage in this since it delays the time before a cell emerges from the constriction portion into the measurement region of the measuring microchannel. In embodiments the constriction portion can be 8 micrometers in width. In embodiments after the constriction portion and before the exit port the at least one microchannel can be about 25 micrometers wide and 25 micrometers in height. In embodiments the height of the constriction portion and microchannel can be at least 20 μηι, at least 25 μηι, at least 30 μηι, at least 40 μηι. It is considered a greater height in the constriction portion will minimise shear stress on the cell

In embodiments the cell receiving portion of the microchannel interposed between the access port and the constriction portion can be shaped to provide a funnel shaped hopper with an access of about 125 micrometers in width at least 20 micrometers in height and about 85 micrometers in length. In embodiments the length can be from the supply microchannel to the constriction portion opposite the access to the hopper. The hopper dimensions should be sufficient to capture a cluster of a minimum of 5 cells. In embodiments, the hopper can have an access with a minimum width of 80 micrometers and a length of 60 micrometers. The height of the hopper may be at least 20 micrometers as a minimum. A height of about 20 micrometers can be advantageous as it minimises blockage of the microchannel by cell clumps. The hopper width and height can extend from the opening of the hopper at the supply channel, for example 125 micrometers in width, and decrease to the constriction portion dimensions, for example to between 5 to 8 micrometers.

In embodiments the microchannel can further comprise a loading reservoir interposed between the access port and the supply channel of the microchannel wherein the reservoir is a void formed in the substrate wherein the void can be capable of holding about 75μ1 of fluid.

In embodiments the supply channel can taper from about 500 micrometers wide at the supply channel end adjacent to the access port of the loading reservoir to about 50 micrometers wide, preferably with a minimum width of at least 20 micrometers and a maximum width of at least 100 micrometers at the end of the supply channel adjacent to and in fluid connection with the cell receiving portion and constriction portion. In embodiments, the supply channel may extend to the access port without increasing in width, i.e. remain about 50 micrometers in width at the access port. The supply channel typically has a minimum height of 20 micrometers to prevent potential blockage by cell clumps. It is considered that 500 micrometers is a maximum initial width of a supply channel as any larger and cell entry due to hydrodynamic flow is very slow. A 50 micrometer width of supply channel adjacent to the cell receiving portion is considered to advantageously minimise 'drift' of cells away from being drawn into the cell receiving portion, for example a hopper or hoppers, due to the creation of potential dead areas of flow and/or a slow flow rate. The minimum dimension of the supply channel adjacent to the receiving portion suitably is no less than 25 micrometers to prevent blockage by cell clumps. A blockage in this area could hinder the correct operation of the device. The width of the supply channel adjacent to the cell receiving portion preferably provides a substantially consistent spacing relative to each hopper such that the hydrodynamic flow at each hopper is identical prior to cell loading. This ensures that as cells are loaded into the hoppers and positioned at the respective constriction portion, the remaining cells are loaded into "free" hoppers such that the hydrodynamic flow equalises the number of cells loaded at each hopper portion.

In embodiments, the supply channel can be orientated at 90 degrees to the cell receiving portion. Orientating the supply channel at 90 degrees to the cell receiving portion simplifies the design of the device to ensure a substantially consistent spacing between the respective hopper entrance and adjacent supply channel portion. By providing the supply channel with consistent spacing to create a width of supply channel, preferably between 25 micrometers and 50 micrometers from the entrance to the respective cell receiving portion (hopper), cells can flow past the opening to the cell receiving portion, rather than enter the cell receiving portion by default. A cell in suspension passing the opening to a cell receiving portion will encounter a force acting to push the cell into the cell receiving portion. Should a cell enter, it will be held at the constriction portion, reducing fluid entry to the measuring channel of that cell receiving portion. Subsequently other cells in fluid suspension in the supply channel, will then experience a lower force pushing them to enter that cell receiving portion and this will cause these cells to move into a further cell receiving portion and in turn measuring channel that has greater hydrodynamic force pushing the cell into a further cell receiving portion until all the cell receiving portions experience equal or substantially equal fluid pressure. The fluid pressure and the equalisation of fluid pressure across multiple cell receiving portions is the controlling feature that locates cells at the constriction portion of each cell receiving portion without user intervention.

In embodiments the exit port of the microchannel can extend to an exit reservoir. The reservoir can be any suitable means to collect fluid which has passed through the microchannel. The device may comprise a plurality of microchannels in fluid connection with one or more access ports. Where a plurality of microchannels are provided, fluid sample may be provided to each microchannel via respective access ports or via a singular access port in fluid connection to a constriction portion adjacent to each of the microchannels.

In embodiments, a supply microchannel can provide fluid suspended cells to several measuring microchannels at the same time wherein, for example the supply microchannel provides fluid sample to at least two cell receiving portions. In alternative embodiments, the supply microchannel can have at least 3 branches, at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, at least 10 branches, wherein each branch provides fluid suspended cells to a cell receiving portion or portions adjacent to a constriction portion at the entrance to a respective measuring microchannel. This is advantageous as it allows multiple measuring microchannels to be used to measure cell migration whilst only requiring the user to provide a fluid sample to a single access port. In embodiments a number of individual migration measurement microchannels, which in use can each contain migrating cells, can be located on the same device to form an array thereby allowing multiple assays to be performed in parallel. Suitably, as each device can be identical, any experiments may be scaled as required over several devices. The necessary control experiments may also be included.

In embodiments the substrate of the device can be optically transparent. Suitably, only a portion of the substrate is optically transparent such that a user can visualise a cell in the measuring microchannel. Alternatively, the substrate or a portion thereof may be transparent to particular wavelengths of light which allow a cell in the measuring microchannel to be tracked, for example by a detector. For example the cells to be measured may be tagged with a fluorescent marker or the like and a detector may be used to monitor the position of the tagged cells in the measuring microchannel.

As will be appreciated, the device can be provided as a disposable or replaceable product or as part of a larger apparatus or system. In embodiments the microfluidic device can be compatible with most common optical microscope systems. In embodiments the device may be prepared by lamination of a number of substrate layers, injection moulding, embossing, precision machining, etching and various other microfabrication and fabrication methods. Lamination of a number of substrate layers may be advantageous to allow the device to be more easily manufactured. Each layer may be elongate and have a length which is greater than a width of a layer and which is greater than a thickness (height) of a layer. Suitably, in embodiments a continuous irreversible seal between the layers, for example first and second layers, is provided to prevent loss of cells or culture media from the microchannels. Suitably the device may be about the length and width of a microscope slide. In embodiments the device can be formed from biocompatible, optically transparent material such as glass, silicone elastomer or other polymeric material. In embodiments, the device can comprise at least two layers, for example a first layer and a second layer. Each layer may be elongate and comprise first and second elongate surfaces along the length and width of the layer. In such embodiments, a first layer can be provided with at least one access port in a first surface of a first layer in fluid connection with an indentation on a second surface of the first layer. During manufacture of the device, the second surface of the first layer can be attached to a first surface of a second layer of the device such that the indentations of the first layer when provided to the second layer effectively provides a hollow microchannel in the substrate.

Suitably, the pattern of indentations may be recessed to a distance of not less than 20 micrometers into the second surface of the device first layer such that when the second surface of the first layer is sealed to the first surface of a second layer an enclosed microchannel network is formed.

As will be appreciated in alternative embodiments a second layer may be provided with indentations in a surface such that when the a first, second or subsequent layer is brought into contact with the surface comprising the indentations, an enclosed microchannel network is formed.

Thus, in embodiments, the microchannels can be recessed into the first surface of the second layer and the first layer is only provided with access ports such that when the first layer is aligned such that the access ports are in fluid communication with the indentations of the second layer, enclosed microchannels are formed. Alternatively, both the first and second layers may include indentations which when aligned form enclosed microchannels.

In embodiments the device can be in the format of a standard microscope slide. Suitably, the device may have the dimensions of about 75mm in length and 25 mm in width. The device may be around 5mm in thickness (height). In embodiments the substrate of the device or a layer thereof can be gas permeable and optically transparent. In embodiments where such a layer of substrate is provided, this can be permanently sealed to a second layer. In such embodiments, the first layer can bear a layout of indented channels of defined size and shape which act to form hollow microchannels when in contact with the substrate of the second layer.

Alternatively, or additionally, the second layer may be provided with indented channels of defined size and shape which act to form hollow microchannels when in contact with the substrate of the first layer.

Suitably the substrate or at least a layer of the device may be rigid.

Preferably the device includes access port closure means, such as a cap to prevent accidental contamination of the device by bacteria or airborne particles and decrease the evaporation of growth media from the device.

In embodiments, the device can incorporate an integrated or embedded measurement scale, visible alongside the measuring microchannel(s) when using an optical microscope. Since, the constriction portion of the devices causes cells to begin migration when they are aligned at the start of the measuring microchannel, quantification of a cell migration distance can be achieved by microscopic visual comparison of a position of a cell in the measuring microchannel relative to a measurement scale located in parallel to the microchannel. This allows each cell's migration to be accurately compared with others. The rate of cell migration can easily be quantified by division of migrated distance travelled by the cell by the time taken to move that distance.

Accordingly, a second aspect to the present invention provides a cell migration measurement system comprising a device according to a first aspect and detection apparatus which is arranged to detect movement of cells in a fluid sample in the cell migration measuring portion of the device. Any detection apparatus may be used which is capable of detecting a cell in a measuring microchannel. The detection apparatus may record images of the cell movement. The detection apparatus may be a microscope, camera or the like. Suitably the detection apparatus may include one or more lens and / or filters and / or mirrors.

The system may further or alternatively comprise positioning apparatus to position the device relative to the detection apparatus. The positioning apparatus may comprise a shaped locating feature, for example a platform, a groove or protrusion to correctly locate the device relative to the detection apparatus. The system may comprise a heater or the like which supplies heat to the device and is capable of regulating the temperature of the fluid sample in the device, for example to regulate the temperature at around 37°C.

According to a third aspect of the present invention, there is provided a method of measuring cell migration, the method comprising the steps:

providing a fluid sample to a microchannel of a device according to the first aspect

moving said fluid sample to a cell migration measuring portion of the device, and

determining the cell migration of cells provided by the fluid sample at a cell migration measuring portion of the device. The device provides for the rapid and straightforward quantification of cell migration. It provides for the alignment of cells at a defined start position at the beginning of the assay. Once this alignment has been established the walls of the measuring microchannel act to constrain the cell migration to a linear path. Any displacement along the microchannel will therefore represent linear cell migration (assuming that no back-tracking of cells has occurred). Advantageously cell alignment is passively self-regulated due to the constriction portion provided at the entrance to the measuring microchannel and requires no involvement from the end user beyond addition of prepared cells in suspension to the microchannel.

Typically the cell receiving portion reaches a defined cell fill level since the hydrodynamic resistance of the hopper and loaded/aligned cells balances the hydrodynamic flow of the cell-transporting fluid stream. The cell receiving portion thus does not over-fill with cells, and when multiple cell receiving portions are provided in the microchannel when a first cell receiving portion (hopper) has reached the determined fill level, the cells can move in the supply microchannel to at least a second cell receiving portion.

Typically an array of ten-measurement microchannels may be provided in parallel. Each hopper in the array receives approximately the same cell load due to the "self regulation" of the cell receiving portions which only accept cells to a defined fill level. The starting environment is therefore as close as possible for each replicate measurement microchannel in the array.

The shape (hopper or funnel shape) of the cell receiving zones ensures that they do not over-fill with cells and minimises underfilling allowing the loading of the measuring microchannel to be passively controlled such that each measuring microchannel contain approximately the same number of cells. By hopper is meant a shaped portion that generally tapers from an access portion towards the constriction portion where cells are discharged by the hopper such that cells undergoing chemotaxis can pass into the cell measuring microchannel. The hopper shape functionally focusses cells provided by the supply channel to the constriction portion to accumulate cells at the constriction portion.

The fluid sample can comprise cell media or other fluids such as, but not exclusively; water, buffer or alcohols. These may be components of cell labelling solutions for example dye or antibody solutions or even cell fixatives such as paraformaldehdye. In embodiments, the step of determining the cell migration of cells provided by the fluid sample at a cell migration measuring portion of the device can be by visualisation, i.e. by looking at a cell as it migrates along the measuring microchannel. Alternatively, an image (s) can be recorded of the cell migrating along the measuring microchannel. When images are recorded, a number of images at different time points can be considered to determine the movement of a cell.

In embodiments it may be possible to enhance the visualisation or detection of a cell in the measuring microchannel by labelling the cells, for example with a colour or fluorescence marker prior to, during, or after the step of providing the fluid sample to the device.

Suitably, where the cells are labelled, the method may include the step of providing a wavelength of radiation which can excite the label.

In embodiments, the method can further comprise the steps of:

- providing a reagent or exogenous compound to the fluid sample;

- moving said reagent in the fluid sample to a cell migration measuring portion of the device;

- determining the cell migration of cells provided by the fluid sample at a cell migration measuring portion of the device in the presence of the reagent or exogenous compound; and

- comparing the cell migration of cells provided by the fluid sample at a cell migration measuring portion of the device in the presence and absence of the reagent or exogenous compound.

Suitably a reagent or exogenous compound may be provided to the fluid sample prior to, during, or after the step of providing the fluid sample to the device. Alternatively, a reagent or exogenous compound may be provided to the device such that movement of the fluid sample within the microchannel causes the fluid to come into contact with the reagent or exogenous compound. In embodiments the method can further comprise the steps of:

(a) using a plurality of time sequential images of cells moving at a cell migration measuring portion,

(b) for each time sequential image, processing the image to identify at least one cell in the image, wherein the same cell is identified for each time sequential image,

(c) determining the position of the at least one cell in the image,

(d) repeating step (c) for one or more successive pairs of time sequential images

(e) determining the cell migration of the at least one cell in the image between time sequential images.

In the case of an embodiment with an embedded measurement scale, only a single image is necessary and it can be assumed all cells begin migration at the zero micrometer distance provided on the scale and so only an image recording the cell's final position is required to measure a cell's total migration.

Advantageously the device and methods of the invention can be used with patient- isolated tumour cells, or indeed another cell type, for example to diagnose particular medical conditions or disease states for example invasiveness of a cancer cell. In such methods cell samples from subjects with diseases or medical conditions, for example cancer, obtained at different time periods can be assayed and results compared. Additionally or alternatively cells can be tested under different test (presence of reagent) conditions. Suitably different test conditions may include the presence of either migration inducers or inhibitors. Dose-dependent increase and decreases in migration using the device and inducers or inhibitors can therefore be tested.

In the use of the device, cells in a fluid suspension are supplied to the internal microchannels of the device. The supply of cells and fluid for example growth medium to the internal microchannels provides a fluid stream extending from the access port to the exit port of the device. Suitably, individual cells may be sequestered from the fluid stream and accumulate at an entrance to a measuring microchannel, which is defined by the cell receiving portion and the constriction portion. Upon sustained incubation of the fluid suspended cells at physiological conditions, the cells at the entrance to the measurement microchannel (constriction portion) undergo a morphological change and can move past the constriction through the passage provided therein and move into the measurement microchannel whereupon the cell migration is effectively constrained to one dimension. This permits a straightforward quantification of individual cell migration by comparison of cell position along the measurement microchannel with an integrated measurement scale located in parallel to the microchannel.

Suitably, in use, cells in fluid suspension may be supplied to a microchannel within the device via an access port in the device using a micropipette. By virtue of the microchannel arrangement at and prior to the constriction portion at the entry to the measuring microchannel, the device itself passively controls the loading of approximately the same number of cells into the constriction portion immediately adjacent and prior to each and every measuring microchannel. The user can therefore be assured that at the commencement of the assay, all cells have begun migrating from the same position and, where multiple measuring microchannels are provided that the microchannels function as replicates of each other.

Suitably, in use, cell culture medium of the fluid sample can form a continuous fluid steam between the access port(s) and the microchannel(s) to the exit port(s), ensuring that physiological conditions persist throughout the interior of the device. The integrated microfluidic channels can allow the fluid requirements of the assay device to be kept well below 1 ml. This is advantageous when working with expensive or difficult-to-source cell treatments. Suitably, in use, the device can provide that the cells may be observed at any time. This allows the progress of migration to be monitored and even recorded throughout the experiment if desired. Advantageously, at the end of the cell migration experiment, the cells may be amenable to further processing. This may include antibody labelling or cell staining using user- reagents supplied via an access port(s) used to supply the cells to the microchannels. This allows the end-user to potentially gain more information from device-localised cells, post-migration.

According to a fourth aspect of the present invention there is provided a computer program comprising instructions for carrying out the method of the third aspect wherein the computer program is executed on a programmable apparatus. Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.

Throughout the specification, unless the context demands otherwise, the terms 'comprise' or 'include', or variations such as 'comprises' or 'comprising', 'includes' or 'including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness.

Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country. Detailed Description of the Invention

Embodiments of the present invention will now be discussed, by way of example only, with reference to the accompanying figures in which: Brief Description of the Figures

Figure 1A illustrates an embodiment of a device comprising access ports, reservoirs and microchannels wherein the device comprises first and second substrate layers wherein the indentations forming the microchannels are recessed on the first substrate layer with the second substrate layer being placed in contact and alignment with the first substrate layer.

Figure IB illustrates an embodiment wherein the indentations forming the microchannels are recessed into the second substrate layer with the first substrate layer being placed in contact and alignment with the second substrate layer such that the ports of the first substrate layer are in alignment with the indentations forming the microchannels of the second substrate layer. The indentations recessed into the second substrate form the microchannels in fluid connection with the access port. Figure 2 illustrates an embodiment of the device in plan.

Figure 3 illustrates an embodiment of the device with ten microchannels provided in fluid communication with a loading reservoir (1) and exit reservoir (6) wherein the supply microchannel (2) tapers from the loading reservoir (1) and forms a hopper configuration and then a constriction portion prior to each of the ten measuring microchannels (4) provided in parallel with the measuring microchannels then forming a fluid exit port (5) and exit reservoir (6).

Figure 4 illustrate an expanded view of a portion of figure 3 wherein the supply channel (2) provides fluid and fluid suspended cells to the cell receiving portion shaped as a horizontal hopper (3) and then to the constriction portion (7) and measuring microchannel (4), alongside which is provided a measurement scale (10), the fluid can then exit the measuring microchannel via the fluid exit port (5).

Figure 5 illustrates a side on view of a microfluidic device of the invention in which the loading reservoir (1) is illustrated in fluid connection with the exit reservoir (6) via a microchannel (including the supply microchannel, cell receiving portion, constriction portion and measuring microchannel) wherein the loading reservoir fluid (30) level is depicted by X and the exit reservoir fluid (40) level is depicted by Y, which levels provide for the hydrodynamic flow through the microchannel and cause the cells (20) to move into the cell receiving portion and chemotactic cells can move along the microchannel (50).

Figure 6 illustrates a graphical representation of the results of an experiment conducted using the device showing the migrated distance of cells in the microchannels labelled A to J.

Figure 7 is an image illustrating fluid suspended cells (60) provided at the hopper formed by the microchannel at a portion of the microchannel forming a constriction. Figure 8 is an image illustrating the changing cell morphology wherein the cells change from roughly spherical to a flattened shape (70) of decreased cross-sectional area enabling them to pass the constriction portion.

Figure 9 is an image illustrating a cell which has passed through the constriction portion and the movement of the cell relative to an integrated measurement scale.

Referring to Fig. 1 the device is preferably constructed from two layers which form first (upper) and second (lower) layers. In the present embodiment of the device the dimensions of the device are those of a standard microscope slide, about 75mm in length and 25 mm in width. The first and second layers have a height no more than 5 mm to 6 mm. The second layer is about 1.5 mm in height. This format is compatible with a wide range of laboratory equipment and will be familiar to most end-users.

The first layer may be constructed from a biocompatible, optically transparent material such as glass, silicone elastomer or other polymeric material. The second layer may utilise the same material. Alternatively dissimilar materials may be used in each layer, provided a means of sealing of one to another is employed.

The first layer bears a pattern of indentation on a second side of the first layer which is in contact with a first side of the second layer. This pattern of indentation can form a reservoir base, supply and measuring microchannel(s). An embedded measurement scale can also be provided.

The pattern of indentations are recessed to a distance of not less than 20 micrometers into the second surface of the device first layer material such that when the first layer is sealed to the second layer an enclosed microchannel network is formed.

As will be understood, the second layer may be provided with indentations to form the microchannels either in addition to or as an alternative to the indentations on the first layer.

Access ports (1), illustrated in figure 1 as circular ports, are provided that fluidly connect to the indented reservoir on the device first layer. These access ports can be on the first side of the first layer opposite that in contact with the second layer. The access ports permit addition of cells suspended in culture media to the microchannel array using a micropipette.

The walls of the access port form a cavity or void in the first layer to hold a reservoir of culture media necessary for maintenance of cell function. The reservoir may have a diameter of not less than 4.5 mm and not more than 6 mm to allow ease of access for the tip of a micropipette yet minimise the surface area for evaporation. In the present implementation of the invention each reservoir holds approximately 75 μΐ of fluid.

Use of the device

In use a fluidic supply of cells is provided via the access port to the loading reservoir and then to the measuring microchannel. Due to the constriction portion at the entrance of the measuring microchannel, the dimensions at the entrance of the measuring microchannel are less than those of a fluid-suspended cell and the fluid suspended cells are excluded from entry into the measuring microchannel. Upon cell maintenance at physiological conditions the cells at the entrance to the measuring microchannel revert from a fluid-suspended (substrate-detached) cell morphology to their substrate-attached morphology. In the substrate-attached morphology the cells can then pass the constriction portion and gain entry to the measuring microchannel where they are constrained to migrate along the length of the measuring microchannel. Migrating cells are at all times in contact with the substrate and / or the sidewall of the microchannel. An embedded measurement scale located parallel to the measuring microchannel may be used to correlate cell position in the measuring microchannel to a real distance in micrometers. Method of Manufacture of Device

In the embodiment of the device illustrated, the reservoirs, supply channels and measuring microchannel array are indented into the second surface of the first layer (half) of the device. This may be achieved by moulding of the second surface of the first layer against a micropatterned master template (a nickel microembossing tool) using PDMS (Polydimethylsiloxane) in a 1:10 ratio of curing agent to polymer. A defined volume of PDMS is used for the entire mould to ensure uniformity of device height between production runs. The PDMS is cured in-situ and then carefully peeled from the mould yielding a micropatterned upper component. Individual devices are cut from the cured PDMS and access ports produced using a sterile 4.5 mm biopsy punch. The second layer (onto which the migrating cells attach) is accordingly a substantially planar substrate either natively biocompatible (such as glass) or, in the case of some polymers (such as polystyrene) rendered biocompatible by prior treatment. The PDMS first layer is treated using corona-discharge before being placed into conformal contact with the second layer (half). After 24 hours a permanent bond is established between first (upper) and second (lower) components, resulting in a completed device.

Alternatively, the device first layer may be constructed from either a polymer such as polystyrene or cyclic olefin copolymer. A hot embossing process with a micro patterned master (nickel microembossing tool) can be used to indent the micropattern into the second surface of the device first layer. Bonding between the first and second layers can be achieved by solvent / heat lamination of a gas permeable film / sheet (<1.5 mm thick) to form the base. This film (onto which the migrating cells attach) can be either natively biocompatible or capable of being treated to achieve a biocompatible surface.

As will be appreciated, a device may be formed wherein the indentations are provided on the second layer and suitably aligned with a port of the first layer. In such embodiments the material selected to form the first and second layers and to provide microchannels will be chosen accordingly.

Cells provided via the access port are transported in a fluid stream from the reservoir to the measuring microchannel by means of a tapered supply microchannel. Referring to the annotations in Fig.3, the beginning of the tapered supply microchannel, abutting the loading reservoir forms the entrance to the measuring microchannel(s). This tapered supply microchannel is 500 micrometers wide and gradually reduces to a 50 micrometers wide microchannel that connects to the measuring microchannels via the cell receiving portion and constriction portion. Where, as in the embodiment illustrated, multiple measuring microchannels are provided, these can be provided as an array to allow the cell movement between measuring microchannels to be compared. The taper of the supply channel provides a gently increasing hydrodynamic flow that imposes order on the cells in transport. This prepares the cells for alignment at the measuring microchannel entrance (see Fig.4 for further microchannel detail).

To obtain representative migration values for the cells under test it is desirable that a number of cells are assayed in identical measuring microchannels at the same time and under the same assay conditions. In the embodiment of the invention illustrated, ten measuring microchannels are arranged in parallel. Referring again to Fig. 3, each microchannel is connected sequentially to the linear portion of the tapered supply channel which in turn is connected to the user-accessible loading reservoir. Addition of fluid to the loading reservoir results in the division of the hydrodynamic flow between individual measuring microchannels of the array before it recombines and exhausts into a common fluid exit port. This common fluid exit port can be in fluid communication with a user-accessible exit reservoir. As illustrated by Fig. 4 each measuring microchannel is preceded by a portion of the microchannel (cell receiving portion) shaped to form a hopper or funnel and designed to hold a cluster or population of cells aligned at the entrance to the measuring microchannel at a constriction portion. Typically, the hopper is 125 micrometers wide where it connects to the supply microchannel and extends 85 micrometers towards the measuring microchannel entrance. The sidewalls of the hopper are angled horizontally such that they form an 8 micrometer wide gap or constriction portion in the microchannel. This constriction portion extends forward into the microchannel for 12.5 micrometers before widening out to match the full 25 micrometer width of the microchannel.

The 8 micrometer passage of the constriction portion is of a smaller size than the mean cross-sectional diameter of a substrate-detached fluid suspended mammalian cell. A vertical height of 20 micrometers (prescribed by the depth of indentation of the microfluidic network into the second surface of the first layer of the device) dictates that the entrance into the microchannel from the hopper forms a vertically oriented rectangular slit - about 20 micrometers tall and 8 micrometers wide. Cell retention is typically achieved since substrate-detached (fluid) cells (in fluid- suspension) exhibit limited plasticity and do not sufficiently deform in the vertical axis to fit through the full height of the constriction. Despite the retention of one or more cells at the hopper-measuring microchannel constriction portion there is still enough vertical space available to allow a decreased volume of hydrodynamic flow into and along the measuring microchannel.

Hydrodynamic flow is the main force (not gravity) that causes the cells in fluid suspension to be located in the respective cell receiving portion (hopper or funnel). Microchannels that have cells loaded within the hopper exhibit decreased hydrodynamic flow. The decreased hydrodynamic flow provides passive regulation of measuring microchannel cell loading. This provides even cell loading into the hoppers. Taking the embodiment illustrated by Fig 3 which has an array of ten parallel measuring microchannels as an example, flow is therefore evenly divided over the ten measuring microchannels and each identical measuring microchannel has equal hydrodynamic resistance. As soon as a cell is retained in one channel the hydrodynamic flow rate within that channel reduces and the total hydrodynamic flow is divided between the other nine microchannels. This further increases the likelihood of a cell being retained at the hopper-measuring microchannel constriction of one of those nine channels. As the loading process continues; driven by the dynamically altering hydrodynamic flow rates, an even distribution of hopper-retained cells emerges over all ten hopper-microchannel interfaces. In the current form of the invention; sufficient room is provided in each hopper to allow the accumulation of several cells. (See figure 7 in which multiple cells have accumulated at the hopper-microchannel interface constriction).

Referring to the side elevation schematic in Fig.5, the hydrodynamic flow that is responsible for cell loading in the hoppers of the microchannel array is generated by the differential height in fluid levels between the two reservoirs. Provided the fluid head height is greater in the loading reservoir than the exit reservoir, a positive hydrodynamic flow will be generated in the interconnecting, supply and measuring microchannels. At the time of supply of substrate-detached cells in fluid suspension to the device (device loading) the difference in fluid head height between the reservoirs should typically lie within the quotient range of 1.25 - 1.6. The quotient value relates the initial set reduction in fluid head height of the exit reservoir relative to the fluid head height of the loading reservoir.

Expression of the fluid head height differential as a quotient allows for an embodiment-independent generation of the optimal hydrodynamic force for the cell types typically used in the invention. The only requirement is that the cross- sectional area and geometry of both the loading and exit reservoirs are known for the particular embodiment under consideration. With this information it can then be established what volumes of fluid need to be added to each reservoir to establish optimal hydrodynamic flow.

Example 1: A device with identical loading and exit reservoirs.

A reservoir with circular cross-sectional area of 12.56 mm ² (4 mm diameter) and vertical sidewalls will exhibit an approximate fluid head height of 2 mm when supplied with 25 μΐ of cell containing fluid. This is obtained from the formula for the volume of a cylinder: volume=nr ² h and assumes that cell volume and internal microchannel volumes are negligible. By dividing the loading reservoir fluid volume by the differential quotient (25 μΐ / 1.25 - 1.6) it can be seen that the optimal fluid volume range required in the exit reservoir is approximately 20 - 15 μΐ.

Example 2: A device with different loading and exit reservoirs.

If the loading and exit reservoirs are of different size then the difference in areas can first be calculated by dividing the area of the loading reservoir by the area of the exit reservoir. To scale the necessary volume of required fluid in the exit reservoir the calculated fluid volume required in the loading reservoir is divided by the value obtained for the difference in areas, that number is then divided by the desired differential quotient. This yields a volume value that should be added to the exit reservoir to establish optimal hydrodynamic flow.

If the quotient value is reduced to below 1.25 the ensuing hydrodynamic flow will likely be insufficient to transport cells from the loading reservoir to the hopper- measuring microchannel interface constriction. Increasing the quotient value would likely lead to an excessive hydrodynamic flow rate which can elastically deform the cells and force them through the constriction between the hopper and the measuring microchannel. This potentially subjects cells to excessive shear stress and prevents correct cell alignment at the hopper-measuring microchannel constriction.

Hydrodynamic flow will continue until it is balanced by the hydrodynamic resistance of the microchannels. This resistance gradually increases during cell loading due to the reduction in cross-sectional area of the hopper-measuring microchannel constriction by accumulating cells (see figure 7). The hoppers cannot be over-filled with cells since before over-filling occurs; the hydrodynamic flow reduces to a level to which it is incapable of moving cells into the supply channel. Upon incubation at conditions to suit the cells under assay (typically for 2 hours at 37°C and 5% C0 ₂ ) cells will attach to the base of the reservoir, supply channel and hopper. This attachment results in a change in the cells morphology from a roughly spherical to a flattened amoeboid shape with a resulting decrease in cross-sectional area (see figure 8). This shape change allows cells to freely pass through the hopper-measuring microchannel constriction and enter the measuring microchannel. Upon sustained incubation cells migrate, forming transient attachments to the microchannel walls and base. A migrating cell maintains close association with the microchannel wall and so will follow the wall continuously. Thus migration within the microchannel is geometrically linear and is effectively constrained to a single dimension (see Fig. 9). Cell migration may be observed using a microscope at any time point over the course of the assay. It is suggested that brightfield magnification in the range of xlOO - x 400 will provide the simplest means of viewing migrated cells. Referring to Fig. 4, this embodiment of the device incorporates a measurement scale aligned a minimum of 20 micrometers below and parallel to each microchannel; to avoid interfering with the seal between the upper and lower surfaces around the microchannel. For ease of quantification this scale has tic marks situated every 50 micrometers along its length. Each 100 micrometer tic is 30 micrometers in height and each 50 micrometers tic is 15 micrometers in height to provide a visual distinction between the indicated distances on the scale. Each 500 micrometer tic is demarcated by the relevant numerals appended underneath for rapid orientation when using the xlOO microscope magnification. (Some variability in tic mark dimensions could be tolerated). To quantify cell migration the position of a defined point of the cell is estimated, relative to the parallel measurement scale. Depending on the particular phase of migration a cell in the measuring microchannel may exhibit a compact or linearly extended form, and can move relatively quickly between the two forms. The defined point used to estimate migration should be established at the commencement of the assay and not deviated from if results are to be comparable between experiments. Rather than rely on the dynamic leading or trailing edges it is suggested that the most accurate point to use for comparison would be the cells centroid position, which can be readily estimated 'by eye' during microscopic examination. The position of the cell centroid should be established relative to the nearest tic on the measurement scale. Given the variability between cell shapes the 50 micrometer resolution of the integrated measurement scale is sufficient to estimate cell position.

Several cells may enter any given microchannel from the hopper population. This is observed particularly over longer incubation times. For the purposes of obtaining data to be used in statistical analysis, the migration distance of the leading cell in each microchannel can be recorded. In this embodiment of the invention up to ten migration values will therefore be recorded per microchannel array. The microchannel arrays may be utilised in triplicate to aid in the establishment of an accurate cell migration average for the cells / treatments being assayed. Three identical arrays can be available on one device for this purpose (see Fig. 2). In the described embodiment of the device the three microchannel arrays are arranged equidistantly along the same horizontal axis to aid in movement from one array to the next when using a microscope X/Y translation stage.

Individual cell migration data may be recorded in tabular format to serve as a permanent record of results. A graph may then be constructed from the tabled data (see Fig.6 for an example scatter plot of the migration of a population of HT1080 Human Fibrosarcoma cells (ECACC no 85111505) within the microchannels of the array). Each point in the scatter plot represents the location of a single cell within the microchannel. Provided the cells have not been assayed for an overly long period (in which the cells may cease to migrate) the point data for the lead cells can be converted into a migration rate by dividing the total distance travelled by the cell by the total time spent migrating. This will yield a value in micrometers per hour (m.p.h) and can be useful when comparing the migration results of different cell lines, or the effect of a particular treatment on migration speed.

Testing of exogenously applied compounds

The described outline has assumed migration of cells in the presence of growth media only. The invention may also be utilised to assay cell migration in the presence of exogenous compounds or reagents. These can be either coated onto the device substrate by prior addition in liquid form, or can be added to the culture medium that surrounds the cells at the commencement of the experiment. Provided that care is taken not to expose cells to excessive hydrodynamic flux, the surrounding fluid, with or without exogenous compounds may also be exchanged during an experiment. By treating cells in this manner it is possible to establish pre and post-treatment migration rates for cells within the migration microchannel array. This ability to quantify and distinguish cell migration distances and rates, in both untreated cells and in the presence of suitable exogenously applied treatments allows the invention to be used as an assay. In conjunction with the proper use of experimental controls this invention is also suitable for establishing the effect on migration of unknown or untested exogenously applied treatments.

Post-Processing of Cells

Cell quantification using this invention relies on visual recording or examination and so is a both non-invasive and non-destructive technique. The fact that the cells remain viable post-quantification allows them to be further characterised if desired.

Subsequent to the filling of the microchannels with fluid and cells a continuous fluid connection is present from the loading reservoir to the exit reservoir. Cells in the measurement microchannels are bathed in and hence accessible to this fluid. It is furthermore possible to add reagents via the reservoirs, provided they are capable of being supplied to the cells via fluid flow. These components can include cell fixing agents, permeabilisation buffers, labelling reagents such as vital dyes, stains or antibodies - to which may be coupled specific labels to aid observation of specific cell features. This further processing may include performing complete further assays or experiments on the cells which are immobilised within the measurement- microchannels via an attachment to the substrate. The first necessary restriction is that experimental reagents are supplied and removed using the microchannels of the device. The second is that results must be obtained indirectly, without direct access to the cells in the microchannels. This could include; for example, the examination of a chemiluminescent or fluorescent label using a suitable microscope.

Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.