WO2009053870A2

WO2009053870A2 - Device and method for the monitoring of the movement of cells

Info

Publication number: WO2009053870A2
Application number: PCT/IB2008/054195
Authority: WO
Inventors: Stefano Cattaneo; Murray F. Gillies; Simone I. E. Vulto; Thomas J. De Hoog; Dennis Merkle; Mark T. Johnson
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2007-10-22
Filing date: 2008-10-13
Publication date: 2009-04-30
Also published as: WO2009053870A3

Abstract

The present invention relates to a device for the monitoring of very small movements of cells, in particular when undergoing chemotaxis. The invention makes use of various ways of electrical detection, like capacitance or impedance measurements. In this way the detection of the movement of cells can be done in situ, which accelerates the chemotaxis assay and increases the accuracy of the measurements. The device according to the present invention particularly comprises a stimulant gradient chamber (1) for providing a concentration gradient of a stimulant, said stimulant gradient chamber (1) having a cell inlet (12) for the inlet of cells (C), - an electrical sensor (2) placed in a measurement area for measuring an electrical parameter which changes in response to a movement of cells (C) in the surrounding of said sensor (2) and for generating a detection signal, and a signal evaluation unit (3) for evaluating said detection signal.

Description

Device and method for the monitoring of the movement of cells

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a device for the monitoring of the movement of cells and to a corresponding method.

BACKGROUND OF THE INVENTION

The mobility of cells in the human body is of vital importance for various biological processes. Cells which can be stimulated to move, often known as chemokinetic or chemotactic cells, include stem cells, leukocytes, fϊbro lasts, tumor cells and of course sperm cells. The cells with the highest mobility are neutrophils which can move at 15-20 μm/ min while the slowest are the fibroblasts which move at only 0.2-1 μm/min.

Monocytes, a type of leukocyte which can chemotax, have many important functions within the human body. One of their most important roles is in the immune system where they are involved in the phagocytosis of foreign cells and materials. Another important property of monocytes is their ability to adhere, penetrate and differentiate at the sites of atherosclerotic plaque formation in arteries. Recently a link between monocyte mobility and atherosclerotic risk has been suggested. More specifically, patients with high risk factors for CAD (such as diabetes and smoking) demonstrate a drastic reduction in monocyte mobility, providing a potential CAD risk stratification based upon monocyte chemotaxis.

These effects are described in more detail in i) Vascular Endothelial Growth Factor- A- Induced Chemotaxis of Monocytes is attenuated in patients with Diabetes Mellitus: A potential predictor for the individual capacity to develop collaterals, Johnnes Waltenberger et al, Circulation 2000; 102; p.185- 190; ii) Hypercholesterolaemia impairs monocyte function in CAD patients, F. S. Czepluch et al, Jour. Of Inter. Medicine 261; p. 201-204 2007; iii) N. Stadler et al: Smoking-induced monocyte Dysfunction is reversed by vitamine C supplementation in vivo, Arteriscler. Thromb. Vase. Biol. 2007;27; p. 120-126.

The traditional method for measuring the mobility of cells such as monocytes is based on a so-called Boyden chamber. Such a device consists of two chambers separated by a thin membrane. The lower chamber is filled with the stimulant (e.g. growth factor), and in the upper chamber the cells are loaded. A chemotactic gradient develops across the thickness of the membrane. After an incubation period the membrane is removed and examined under a microscope to count the number of cells that had sufficient mobility to traverse the membrane. Boydon chamber chemotaxis assays are actually very inefficient assays with only about 10% of the initial sample liquid being used.

The movement of cells can particularly be chemotactic, chemokinetic, chemo invasion or haptotaxis. In chemotaxis the movement of cells is induced by a concentration gradient of a soluble chemotactic stimulus. In haptotaxis the movement of cells is induced by a concentration gradient of a substrate-bound stimulus. In chemoinvasion the movement of cells into/through a barrier or gel is induced by a concentration gradient of a chemotactic stimulus. With chemokinetic the movement of cells happens under a concentration of a chemical but where no gradient is present.

The problem with the known method of measuring the cell mobility is three fold. Firstly, it takes a long period of time for the cells to migrate through the membrane thus the assay is time consuming. Secondly, it is labor intensive to count the number of cells present on the membrane. Finally, the Boyden chamber assay only yields the total number of cells that crossed the membrane after a certain time. Cells which migrate through the membrane in the beginning of the incubation time are not distinguishable from cells which migrate later in time.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a device and method which allow to perform time-resolved measurements of cell migration, e.g., to study the distribution of chemotaxis over a cell population or to study the movement of the same cell under various conditions (i.e. different concentration gradients, after drug treatments etc.).

The object is achieved according to the present invention by a device as defined in claim 1 comprising: a stimulant gradient chamber for providing a concentration gradient of a stimulant, said stimulant gradient chamber having a cell inlet for the inlet of cells, - an electrical sensor placed in a measurement area for measuring an electrical parameter which changes in response to a movement of cells in the surrounding of said sensor and for generating a detection signal, and a signal evaluation unit for evaluating said detection signal. The object is further achieved by a corresponding method as defined in claim 23.

The device and method of the present invention allow to measure the mobility of thousands of cells simultaneously and the distribution of the mobility. By the invention, the time needed to perform the assay is considerably reduced. The invention is based on the idea to integrate an electrical sensor into the stimulant gradient chamber, by which a change of an electrical parameter can be measured. Said parameter is selected such that it is influenced by the movement of the cells to be monitored, so that the movement of cells in the surrounding of the sensor (i.e. in the monitoring area or sensing area of the sensor) causes a change of said parameter. Such changes are reflected in the detection signal, and the signal evaluation unit is adapted for detecting such changes and for outputting information about the movement of the cells.

The structures can be either electrodes or active components such as transistors which sense the position of the cells. By the integrated sensor within a stimulant gradient chamber an assay, e.g. a chemotaxis assay, and the detection is done in situ, which will accelerate the total assay time (a standard chemotaxis assay time is 3-4 hours, from which the chemotaxis process itself takes 1-2 hours). In addition, this will enable the measurement of chemotaxis in cells such as monocyte cells which should be measured within 1-2 hours of the sample being taken. Further, with in situ detection, the detection at the end (like with the Boyden chamber) is not needed anymore. Therefore, less devices handling and less man hours are needed. Still further, the results will be more accurate, since the read out is always done in the same way, the detection process can be easily automated, and statistical information can be obtained directly.

An important physical effect for the present invention is dielectrophoresis (DEP). This effect is observed if particles (such as cells) are placed in a medium with a significantly different permittivity (such as water). If a non-homogeneous electrical field is then applied the cells experience a force which, depending on the frequency of the field, can either drive them to the low field or the high field regions. There are many publications about the details of DEP (e.g. J. Voldman, Electrical Forces for microscale cell manipulation, Annu. Rev. Biomed. Eng. 425-453, 8 (2006)); however, for this invention it is important to realize that in a high conductivity medium (such as PBS and blood) usually only negative DEP is possible (at reasonable frequencies), i.e. cells will avoid areas of high electrical field. Preferred embodiments of the invention are defined in the dependent claims. It shall be noted that the claimed method can be developed further in the same or similar way as defined in the dependent claims of the claimed device.

According to a preferred embodiment the electrical sensor is adapted for measuring a capacitance, resistivity and/or conductivity, in particular by measuring a current, a voltage and/or permittivity. For instance, standard methods for measuring a capacitance can be employed according to the invention.

A simple embodiment of the sensor comprises at least two electrodes located on the same substrate or opposite to each other on opposite substrates, e.g. located in parallel. Having electrodes on the same substrate makes fabrication easier, while electrodes on opposite substrates generally results in a better sensitivity.

Further, a plurality of electrodes arranged in the form of a matrix can be used for monitoring the movement of a plurality of cells simultaneously. Said plurality of electrodes preferably comprises a common reference electrode, the spacing between said common reference electrode and the other electrodes being provided for being filled with cells and concentration gradient of stimulant. Said plurality of electrodes are connected to the evaluation unit via switching elements, in particular active electronic switching elements, for individually measuring the detection signals of the individual electrodes. Preferably, the sensor can be addressed and their detection signals measured individually or in groups, for instance allowing subsequent measuring of the detection signals from sensors of different rows or columns.

The spacing between the electrodes and/or the width of the electrodes is preferably adapted such that a cells fills a substantial portion, in particular at least 30%, preferably at least 50%, of the volume between the electrode to achieve a large sensitivity. In a preferred embodiment the substrate carrying the electrodes is made of a material having a low permittivity, in particular a material having a permittivity below 20, preferably below 10, for instance a plastic material having a permittivity of 3-4. In this embodiment, cells with high permittivity (e.g. water based cells having a permittivity of around 80) will result in a large change of capacitance between the electrodes. In an advantageous embodiment the sensor comprises a transistor or a metal- insulator-semiconductor diode and adhesion promoting molecules arranged on the gate electrode of said transistor or an electrode of said diode, respectively, for capturing cells, indicated by a change of one or more electrical parameters of said transistor or diode, respectively. If the cells are captured by the adhesion promoting molecules the electrical characteristics of the transistor or diode, respectively, shift, which shift indicates the presence of cells on the electrode being provided with said adhesion promoting molecules.

In a further embodiment a gas inlet is provided for the inlet of gas and/or air, in particular of gas or air bubbles, into the measurement area prior to measurement. In this way a larger change in capacitance can be achieved. After the measurement the gas/air is retracted and the liquid flow is resumed.

Different advantageous embodiments of the device of the present invention, in particular of the stimulant gradient chamber, are defined in claims 11 to 22.

In most microfluidic assays for measuring chemotaxis the cells are loaded into the observation area with a pipette or through a microfluidic inlet. This often results in an ill- defined start situation with the cells spreading over a significant section of the monitoring chamber. An ill-defined start position is disadvantageous since with the in situ detection method of the present invention it is sometime difficult to distinguish between cells that moved to a certain position due to e.g. chemotaxis, and cells that had an initial "head start". Further, cells at different locations across the channel experience different conditions.

Hence, in a further embodiment as defined in claims 12 and 13 this problem is avoided by introducing the cells into the stimulant gradient chamber via one of the inlets together with either the buffer or stimulant, preferably by introducing the cells via the buffer inlet. To be able to transport the cells, a stronger fluid flow than that used to generate the concentration gradient is preferably applied to the device. Barrier electrodes (preferably two electrodes) are preferably arranged at each junction in the microfluidic branch structure. Preferably, the electrode elements of the barrier electrodes are driven with sine voltages, one with a phase lag (preferably 180°) with respect to the other, so that an area of high electric field is created in the gap between these electrodes. The cells cannot cross this electrical barrier, so that they are effectively always directed to one the branches on one side of the branch structure. The liquid, however, is not affected by the electrical barrier and can flow equally in both directions at the junctions.

This embodiment makes use of electrical fields thus simplifying the filling of the device with sample cells and creating a well defined begin-state for an assay. The electrical fields can also be used to decrease the wait time required before cell adherence and polarization.

In further embodiments such electrical barriers are provided alternatively and or in addition within the monitoring chamber to provide a well defined starting position of the cells with the monitoring chamber. This embodiment can also be used in other embodiments of the stimulant gradient chamber and is not limited to the use with a branch structure.

In another embodiment so called snake electrodes are provided alternatively and or in addition at the inner walls within the branches of the branch structure to avoid adherence of the cells to the inner walls.

It shall be noted that the embodiments using electrical barriers and/or snake electrodes are not only usable in combination with the electrical sensor proposed according to the present invention, but can also be used in other devices and methods for monitoring of the movement of cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be explained in more detail with reference to the following exemplary non-limiting drawings in which

Fig. 1 shows a schematic block diagram of the general layout of a device according to the present invention,

Fig. 2 shows a first embodiment of a device according to the present invention,

Fig. 3 illustrates standard capacitive measurement used in an embodiment according to the present invention,

Fig. 4 shows different embodiments of an electrical sensor for capacitive measurement,

Fig. 5 shows a schematic block diagram of a first embodiment of a stimulant gradient chamber according to the present invention,

Fig. 6 shows a schematic diagram of a first embodiment using an active matrix layout, Fig. 7 shows a schematic diagram of a second embodiment using an active matrix layout,

Fig. 8 shows another embodiment of an electrical sensor for capacitive measurement,

Fig. 9 shows a schematic diagram of an embodiment using resistivity measurement,

Fig. 10 shows a schematic block diagram of a second embodiment of a stimulant gradient chamber according to the present invention,

Fig. 11 shows a schematic block diagram of a third embodiment of a stimulant gradient chamber according to the present invention, Fig. 12 shows a schematic block diagram of a fourth embodiment of a stimulant gradient chamber according to the present invention,

Fig. 13 shows a schematic diagram of an embodiment using haptotaxis,

Fig. 14 shows a schematic diagram of an embodiment using chemoinvasion, Fig. 15 illustrates the normal start position, the end position and a preferred start position of cells in the monitoring chamber,

Fig. 16 illustrates different conditions influencing the cells at different positions in the monitoring chamber,

Fig. 17 shows a schematic diagram of an embodiment using barrier electrodes in the branch structure,

Fig. 18 shows a schematic diagram of an embodiment using snake electrodes within the channels of the branch structure,

Fig. 19 shows a schematic diagram of an embodiment using electrodes in the monitoring chamber, Fig. 20 shows a schematic diagram of an embodiment using electrodes in the monitoring chamber having a side inlet for cells, and

Fig. 21 shows a schematic diagram of an embodiment having a cell inlet in a branch of the branch structure.

DETAILED DESCRIPTION OF THE INVENTION

Fig. 1 shows a schematic block diagram of the general layout of a device according to the present invention, which can, for instance, be used in a chemotaxis assay. The device includes a stimulant gradient chamber 1 for providing a concentration gradient of a stimulant over the areas where the cells C to be monitored are located. To create the concentration gradient, the stimulant gradient chamber 1 generally comprises a stimulant inlet 10 for the inlet of the stimulant (e.g. a growth factor), a buffer inlet 11 for the inlet of a buffer solution and a cell inlet 12 for the inlet of cells C to be monitored into the inner volume 13 of the chamber 1. There are various ways of providing a concentration gradient of a stimulant. These will be explained below in connection with various embodiments of the invention.

An electrical sensor 2 (or, preferably, an array of electrical sensors 2) for detecting changes of an electrical parameter caused by the movement of cells C in a sensing area (i.e. the area where the sensor 2 is able to detect such changes) is located within the chamber 1. The sensor provides a detection signal and outputs it to an evaluation unit 3 for evaluation. When a stimulant concentration is present in the chamber 1, the cells will attempt to migrate in the direction of the gradient. During this attempt at migration the detection signal is monitored with the evaluation unit 3, which evaluates/processes said detection signal and detects changes in said signal and outputs an information about the movement of cells in the sensing area based on detected changes.

The electrical sensor 2 is only schematically shown in Fig. 1, particular embodiments thereof are shown and explained in more detail below.

Fig. 2a shows a schematic block diagram of a first embodiment of a device according to the present invention. In this embodiment the stimulant gradient chamber 100 includes a branch structure 101 and a monitoring chamber 102. The branch structure 101 has a plurality of interconnected branches (also called micro fluidics) for mixing the stimulant and the buffer solution provided via the stimulant inlet 10 and the buffer inlet 11, respectively, into said concentration gradient of said stimulant. Via a concentration gradient outlet 103 the generated concentration gradient is provided to a concentration gradient inlet 104 of the monitoring chamber 102, into which the cells are loaded via the cell inlet 12. Within said monitoring chamber 102 the electrical sensor 2 is arranged.

Fig. 2b shows a side view of the measurement chamber 102 in which the flow F of the liquid containing the growth factor, the cells C and the gradient G of growth factor are indicated. In preferred embodiments the sensor 2 is adapted for capacitive measurement, preferably standard capacitive measurement. In the situation where a larger change of capacitance may be expected, traditional methods of capacitive measurement are used.

Standard methods to measure a capacitance include: measuring the increase in the voltage V of the unknown capacitance Cm when a known and constant current I is applied as indicated in Figs. 3a and 3b; measuring the voltage division across the unknown capacitance Cm and a reference capacitance Cref when a voltage pulse Vprobe is supplied. It is possible to determine the capacitance by applying a probe voltage pulse to the reference capacitor situated parallel to the measurement electrode of the sensor 2, as illustrated in Fig. 3c. The ratio of the capacitors Cm, Cref determines the height of the voltage pulse Vm at the electrode. In this way the capacitance Cm can be determined.

In embodiments employing such a standard capacitive measurement, the device would comprise at least one set of electrodes forming the sensor 2 positioned so as to probe the capacitance of the area where cells are to be found. For instance, in a first embodiment of a sensor 2A shown in Fig. 4a both electrodes 20, 21 are situated on the same substrate 22 of the sensor 2A, preferably in the form of parallel electrodes spaced by a distance similar to the cell size. Having the electrodes 20, 21 only on one substrate 22 makes fabrication easier and cheaper. By using narrow electrodes placed close to each other, the relative sensitivity to the cells is improved (as the background capacitance decreases).

In a second embodiment of a sensor 2B as shown in Fig. 4b, the electrodes 20, 21 are situated on opposite substrates 22, 223 of the sensor, preferably in the form of parallel plate capacitor electrodes. By keeping the spacing of the parallel plates to a minimum value, the relative sensitivity to the cells is improved (as the background capacitance is even further decreased whilst the capacitive signal is larger). In other embodiments the plates can even be closer together than the diameter of the cell. This then requires the cell to deform and closely resembles the in-vivo situation in the microvascular.

Preferably, in these embodiments the substrate(s) (and/or spacer layer(s) provided between the substrate(s) and the electrodes) has a low permittivity, in particular below 20, preferably below 10. For instance, a typical plastic material is used having a permittivity of 3-4. The cell with high permittivity (water based, around 80) will then result in a large change of capacitance.

A change of capacitance can also be achieved by removing the buffer in which the cells are situated at the moment of capacitance measurement. This is achieved by introducing gas/air bubble (bubble of gas and/or air) to the measurement chamber 102 prior to measurement. An embodiment having a gas/air inlet 13 (e.g. from a gas/air storage - not shown) is shown in Fig. 5. In this manner, an extremely large change in capacitance can be realized. After measurement, the gas/air bubble is retracted and the liquid flow resumed. In this embodiment only one volume with gas is required so that the gas can be moved over the cell volume. Rather than gas or air, it is also possible to use a biocompatible oil with low electrical permittivity. This oil could be moved to be above the cells for a measurement via the principle of electro -wetting.

Advantageously, a multiplicity of electrodes is provided in the device in the form of an array or a matrix of electrodes. In this manner, it is possible to detect a multiplicity of cells at different positions across the device (simultaneously or subsequently).

In a preferred embodiment as shown in Fig. 6 it is proposed to use an active matrix based array of electrodes as the basis of a device for the monitoring of the movement of cells. In an active matrix device, it is possible to re-direct a measuring signal from one driver to a multiplicity of electrodes, without requiring that each electrode is connected to the outside world by a contact terminal.

There are several options for configuring the device depending upon the desired electrode layout. For instance, a discrete array of electrodes can be used, that can be driven by active matrix principles. The device is completed with a second substrate, comprising at least one electrode, the space between the substrates being filled with the cells and growth factor. Preferably, the second substrate consists of just a single common electrode (parallel plate capacitor layout).

An active matrix principle, which switches at each electrode, is advantageous as it reducing the line and row capacitance and therefore allows more accurate measurement of the capacitance which originates from cells.

In the following, the embodiment of a device according to the invention using a plurality of cell measurement devices (CMD, i.e. sensors) driven by active matrix principles as shown in Fig. 6 is explained in more detail. In this embodiment a series of CMDs 2 (in particular their cell measurement electrodes 20) is connected to a series of measurement terminals (i.e. measurement driver terminals 24 and select driver terminals 25) making use of an active matrix to ensure that all cell measurement devices 2 can be independently accessed. The capacitance can be measured by either a voltage or a current measurement signal (as explained above with reference to Fig. 3), and these are connected to the matrix of measurement terminals 24, 25 via (preferably active electronic) switching elements 26 (e.g. transistors, diodes or MIM diode devices). Preferably, electrodes 20 are separate cell measurement electrodes and electrodes 21 are a common electrode, but can also be separate electrodes.

The operation of the device shown in Fig. 6 for independent measurement by a single CMD 2 is as follows. In the non-addressing state, all select lines 27 are set to a voltage where the switches 26 are non-conducting. In this case, no CMDs 2 are being measured. To read a given CMD 2, the switches 26 in the entire select line 27 incorporating the required CMD 2 are switched into the conducting state (by e.g. applying a select signal). The measurement signal (e.g. a voltage or a current) in the column 28 where the CMD 2 is situated is passed through the switch 26 to/from the CMD 2. The select signals of all other rows will be held in the non-select state, so that the other CMDs 2 are attached to the same column 28 via non-conducting switches 26 and will not be measured. After the CMD 2 is measured, the switches 26 in the line are again set to the non-conducting state, preventing further measurement of the CMD 2. The device will then remain in the non-addressed state until the following measurement is required, at which point the above sequence of operation is repeated.

It is also possible to measure more than one CMD 2 in a given row simultaneously by applying a measurement signal to more than one column 28 in the array during the select period. It is possible to sequentially measure CMDs 2 in different rows by activating another row (using the select driver 25) and applying a measurement signal to one or more columns 28 in the array.

In another preferred embodiment, as shown in Fig. 7, the active matrix cell measurement device is realized using thin film transistor (TFT) technology to ensure that all CMDs 2 can be independently driven. TFTs, which are used as switching elements 26 in this embodiment, are well known switching elements in thin film, large area electronics, and have found extensive use in e.g. flat panel display applications. Industrially, the major manufacturing methods for TFTs are based upon either amorphous-Si (a-Si) or low temperature polycrystalline Si (LTPS) technologies, whilst other technologies such as organic semiconductors or other non-Si based semiconductor technologies (such as CdSe) can be used.

Whilst offering somewhat less flexibility than using TFTs, it is also possible to realize an active matrix based cell measurement device using the technologically less demanding thin film diode technology or metal-insulator-metal (MIM) diode technology. Preferably, the electrodes 20 used for measuring the capacitive variation in the embodiments shown in Figs. 6 and 7 may at a later stage be used during the assay to e.g. manipulate the cells or lyse the cells electrically. As shown in Fig. 7, the electrodes 21 are a common electrode connected to ground.

In the situation where a smaller change of capacitance may be expected, it may be advantageous to measure the complex relative permittivity ε_r

ε_r = ε' -jε"

with ε' the real part and ε" the imaginary (out of phase) part of the permittivity. Some methods to measure ε" include measurement of the dissipation factor (ε'V ε') using an impedance analyzer (i.e. an LCR meter or similar), measurement of the RF signal loss along a transmission line using e.g. an impedance analyzer or an oscilloscope and measurement of the deformation of a pulsed signal using e.g. an oscilloscope. In the above explained examples, the active matrix simply acts as a method of routing the probe and measurement signals to and from the measurement electrodes.

Next, an embodiment of the invention using transistor based surface capacitive measurement shall be explained. In the biosensor literature a transistor (or a metal-insulator- semiconductor diode) based capacitive measurement has been described, e.g. in P. Estrela et al, Biosensors and Bioelectronics, 2005, 20, 180. Therein, the gate electrode of the transistor is used as a probe electrode, covered by adhesion promoting molecules, for the sample to be detected. As molecules from the sample are captured by the adhesion promoting molecules, the electrical characteristics of the transistor shift, showing that molecules have been attached to the surface.

The basic idea of using molecules attached to the gate electrode (probe electrode) of a transistor (or an electrode of a diode), which is used as a sensor is used in a device according to the present invention enabling to sense the presence of a cell on the probe electrode. The electrodes could be coated with extra cellular matrix, collgen, fϊbronectin or VCAM . Such a sensor 2C is schematically shown in Fig. 8. Normally a gate is covered with a metal electrode to which a voltage can be applied. Here in this embodiment there is no metal on the gate 30, but molecules 31, and the voltage is applied via the culture medium. If there are cells C on the gate 30 then this forms a resistance and the voltage under the cell is lower, i.e. the gate is not as closed. The current through the transistor 2C can then be used to say how much of gate is covered by cells.

Preferably, VCAM (Vascular Cell Adhesion Molecules) are provided on the gate that is used as starting position. This will locally attach cells, i.e. the cells should still be able to move from the start position.

Now, an embodiment will be explained using resistivity/conductivity measurements. There are many approaches to determine resistivity. For example, by simply measuring a current flowing between two electrodes on a sensor (using a current amplifier) it would be possible to determine the resistivity.

In US 6,301,500, an active matrix electro-stimulation device is described which incorporates a system for measuring the local electrical impedance. Fig. 9, which basically corresponds to Fig. 2 of US 6,301,500 shows a schematic representation of an embodiment of this device. For a detailed explanation of this device reference is made to this document, in particular of the embodiments shown there in Figs. 1 and 2, which is herein incorporated by reference. In the embodiment shown in Fig. 9 the counter electrode 41 is arranged as a conducting strip which meanders between the electrode pads 40. Although only a single meandering strip is shown in Fig. 9, in practice several counter electrodes may be provided between the electrode pads and/or one or more straight conductive strips may be used as counter electrodes.

It is proposed here to detect the presence of a cell (at electrode 40 in Fig. 9) using the change in resistance between the electrode 40 and the counter electrode 41 (which serves as reference electrode). Again, if the cell is in a water based fluid, the change in resistance will be relatively low. For this reason, in a preferred embodiment, the flow of liquid is temporarily stopped during the resistance measurement, and a gas/air bubble is introduced to the measurement area (as is shown in Fig. 5). In this manner, an extremely large change in resistance will be realized. After the measurement, the gas bubble is retracted and liquid flow resumed.

A schematic block diagram of a second embodiment of a stimulant gradient chamber 110 according to the present invention is shown in Fig. 10. In this embodiment the stimulant gradient chamber 110 comprises a stimulant chamber 111 having a stimulant inlet 10 for the inlet of the stimulant, a buffer chamber 112 having a buffer inlet 11 for the inlet of the buffer solution, a plurality of channels 113 connecting said stimulant chamber 111 and said buffer chamber 112, a tapping means 114 for tapping said channels 113 at different tapping locations 115, a concentration gradient outlet 116 for the outlet of the generated concentration gradient, and a monitoring chamber 117 having the cell inlet 12 and a concentration gradient inlet 118 for the inlet of said concentration gradient. Again, within said monitoring chamber 117 the sensor 2 is arranged.

This embodiment is based on the idea of connecting two volumes 111, 112 together via many narrow channels 113. By filling one volume 111 with stimulant and the other 112 with buffer a gradient in stimulant concentration is established along the channels 113. By tapping these channels 113 at locations 115 which start near one volume 111 and progressively move towards the other volume 112 it is possible to create a gradient at the outlet 116 where the monitoring chamber 117 is situated. Fig. 11 shows a schematic block diagram of a third embodiment of a stimulant gradient chamber 120 according to the present invention. In this embodiment the stimulant gradient chamber 120 again comprises a stimulant chamber 121 having a stimulant inlet 10 for the inlet of the stimulant, a stimulant outlet 15, and a buffer chamber 122 having a buffer inlet 11 for the inlet of the buffer solution and a buffer outlet 16, which are connected by a diffusion chamber 123 for creating the gradient in stimulant concentration by diffusion of the stimulant and the buffer solution into the diffusion chamber 123. Within said diffusion chamber 123 the sensor 2 is arranged.

Fig. 12 shows a schematic block diagram of a fourth embodiment of a stimulant gradient chamber 130 according to the present invention. In this embodiment a local gradient in stimulant is provided by defining micro fluidic channels 131, 132 on top of an array of sensors 2. The walls 133 of these channels 131, 132 are slightly porous to aqueous solutions. An example of such a material is a gel type material (preferably a hydrogel type material) such as polyacrylamide. Preferably the polyacrylamide would be photosensitive to allow direct photolithography to define the structures.

By filling alternate channels with stimulant and buffer solution, e.g. channels

131 with stimulant and channels 132 with buffer solution, it is possible to create a local gradient as illustrated schematically in Fig. 12. An added advantage of such channels 131,

132 is that it also aids the filling of the array with cells. With a liquid flow along the direction defined by the channels 131, 132 the cells are transported to the sensors 2.

The hydrogel material that can be used in this embodiment can be based on e.g.: i) Anionic monomers: e.g., (meth)acrylic acid, p-styrene sulfonic acid, itaconic acid (CH2C(COOH)CH2COOH), crotonic acid (CH3CHCHCOOH); ii) Cationic monomers: e.g., vinyl pyridine, aminoethyl (meth)acrylates, acrylamide; iii) Neutral monomers: e.g., hydroxyethyl (meth)acrylate and vinyl acetate.

In the embodiment shown in Fig. 12 hydrogel channels 131, 132 are proposed which would allow stimulant to passively diffuse into the volume containing the sensors 2. This is possible if there is a flow of solutions through the channels 131, 132. However, it may be preferable to avoid the need for flow and create chambers where stimulant and buffer solution are statically present. In this case the volumes will become depleted as the stimulant and buffer solution diffuse away and so the gradient will diminish. It is therefore advantageous to be able to actively control the diffusion into the monitoring chamber. To enable this, in a further embodiment electrodes 135 are placed under the hydrogel walls and the hydrogel is made responsive to either an applied voltage or an associated pH change. Upon applying a voltage to said control electrodes 135 via a control voltage supply 134 the porosity of the walls 133 can be increased in order to compensate for a depletion of the stimulant. In a further embodiment, a gel is provided on top of the sensors to define the distance to the sensors upon adherence and hence make the electrical measurement more accurate.

Fig. 13 shows a schematic diagram of an embodiment using haptotaxis. In haptotaxis the movement of cells is generally induced by a concentration gradient of a substrate-bound stimulus. In this embodiment a collagen layer 40 with cross-linked VEGF (vascular endothelial growth factor, which is an important signaling protein involved in both vasculogenesis and angiogenesis) is provided on top of a carrier layer 41, which carriers the sensor 2 on its bottom. The concentration in bound growth factor can be realized in various ways: i) ink jet printing of growth factor solution where the number of drops (and so also of the concentration) increases; ii) use of photosensitive gel which binds to growth factor and has a gradient illumination. The device is generally working in the same way as the above described embodiments for chemotaxis.

Fig. 14 shows a schematic diagram of an embodiment using chemoinvasion. In chemoinvasion the movement of cells into/through a barrier or gel is generally induced by a concentration gradient of a chemotactic stimulus. In this embodiment the stimulant gradient chamber 50 has walls 51 made of gel, which is porous to the stimulant and buffer and which separates the stimulant gradient chamber 50 from the neighboring buffer chamber 52 containing the buffer solution and the neighboring stimulant chamber 55 with fluid containing growth factor. By diffusion through the gel barriers 53 the gradient is obtained in the stimulant gradient chamber 50. The gel should be optically transparent and offer no electrical resistance. Side walls 53 are provided on the outer sides of the chambers 52 and 55. The chambers 50, 52 and 55 and the side walls 53 are provided on a substrate layer 54 (e.g. a glass substrate), which carriers the sensor 2 on its top surface. The operation of this embodiment is generally the same as explained above for the embodiments using chemotaxis. In the above described embodiments, particularly the embodiments shown in

Figs. 2, 5, 10, the cells are injected from the side inlet 12. This results in a liquid flow in the direction where there will later be chemotaxis and in turn results in an ill-defined start situation such as that shown in Fig. 15a. The situation after the chemotaxis assay is shown in Fig. 15b. The preferred situation with aligned cells, in a pre-defined start position, is shown schematically in Fig. 15c.

Further, these embodiments require two pumps, i.e. a first pump for the buffer inlet 11 and the stimulant inlet 11 (both can be driven from a double syringe pump) and a second pump for introducing the cells via the side inlet 12.

Still further, the cells require an adhesion period in the device before chemotaxis can begin. This may be a significant period of time but is directly related to the cellular chemotactic potential.

An ill-defined start position is disadvantageous for several reasons: With an external microscope, it is possible to track individual cells and reconstruct their movement with tracking software. However, with in situ detection methods based on e.g. electrical measurements (impedance/capacitance/resistance) it is more challenging to track individual cells. Often, these methods cannot discriminate between cells that moved to a certain position due to chemotaxis, and cells that had an initial "head start". Cells at different locations across the channel experience different conditions, including i) A different slope of the concentration gradient (Fig. 16a), if the gradient is not perfectly linear (which is difficult to achieve in practice). Diffusion-based gradients, for example, always have a sigmoidal profile such as that shown in Fig. 16a. ii) A different offset concentration of chemoattractant (Fig. 16b), even assuming an ideal linear gradient. Cells have been shown to react not only to concentration gradients (chemotaxis), but also to changes in the average concentration (chemokinetic). iii) In case of flow-based devices, different liquid flows (Fig. 16c). In the most common case of pressure-driven devices, for example, the flow profile is parabolic, with the highest velocity in the middle of the channel and the lowest velocity at the channel walls. Different flows result in different force components in the direction of the flow and in different amounts of shear stress on the cells, which is known to affect chemotaxis.

In order to avoid the side inlet of the above described embodiments it is suggested to introduce the cells C via one of the inlets together with either the buffer solution B or stimulant S, preferably together with the buffer solution via the buffer inlet 11. A first embodiment of a stimulant gradient chamber 140, which is based on the embodiment shown in Fig. 2, is schematically shown in Fig. 17a comprising a branch structure 141 and a monitoring chamber 142. To be able to transport the cells, a stronger fluid flow than that used to generate the concentration gradient is preferably applied to the device. It is further suggested to situate electrode barriers 143 (preferably two electrodes) at the junctions in the micro fluidic branch structure 141. The exact positioning of the electrode barriers (as necessary at the first junction) is shown schematically in Fig. 17b.

When the electrodes of an electrode barrier 143 are driven with sine voltages, one with a 180° phase lag with respect to the other, then an area of high electric field is created in the gap between these electrodes. The cells enter via the buffer inlet 11, but since they cannot cross the electrical barrier 143, they are effectively always directed to the left in figure 17a. The liquid, however, is not affected by the electrical barrier 143 and can flow equally in both directions at the junction. This embodiment is not limited to the use of two electrodes for the barrier electrodes 143, as multiple electrodes (with multiple phases) can also be used, for example, to introduce traveling wave dielectrophoresis. It is also possible to situate electrodes on both top and bottom substrates to create a more effective field barrier. Electrodes should at least be placed at each branch of the tree on the far left side in the embodiment shown in Fig. 17a, if it is desired to position the cells C on one side of the gradient, although electrodes at every junction would allow cells not only to be switched to the side with the lowest concentration of growth factor but also to set up any other distributions of cells in the monitoring chamber 142. After the cells have reached the monitoring chamber 142, the fluid flow will be reduced to a level that does not disturb the chemotaxis assay, but is still sufficient to create a stable concentration gradient.

Another embodiment avoiding a side inlet for the cells is schematically depicted in Fig. 18. Since monocyte cells are adherent there is a possibility that introducing the cells via the buffer inlet 11 may result in the cells adhering to the inner walls of the branch network 151 of the stimulant gradient chamber 150 (cf. Fig. 18a). In order to avoid this long electrodes or "snake electrodes" are placed at the inner walls along the entire length of at least channel 153 of the branch structure 151, through which most or all of cell will flow (in Fig. 18a the left-hand channel). Of course, all other channels of the branch structure 151 can also be provided with such "snake electrodes".

A cross-section of the channel 153 is shown in Fig. 18b. As can be seen, in this embodiment four snake electrode elements 160-163 are arranged in the areas of the corners of the channel 153, in particular on the glass substrates 164, 165 close to the chamber walls 166, 167. By driving the electrodes 160-163 with the voltages as depicted in Fig. 18b, in particular by driving the electrodes 160, 163 with a sine wave voltage having a phase lag of substantially 180° with respect to the drive voltage of the other two electrodes 161, 162, the cells C are held in the middle of the channel 153.

Similarly as with the above shown embodiment (Fig. 17), the fluid flow will be increased to transport the cells and decreased to perform the chemotaxis assay (e.g. by a control unit controlling the respective pumps). Such an embodiment can be applied to various microchambers (i.e. not just chemotaxis assays) in order to prevent nonspecific cell adhesion to the walls of a device.

As was mentioned previously, the starting position in traditional chemotaxis assays is often poorly defined. This problem may already be alleviated by introducing cells via the buffer inlet as was suggested in the previous embodiments. Since there is laminar flow in the monitoring chamber 152 the cells will distribute along the direction of the flow which is now orthogonal to the growth factor gradient direction. This is in contrast to the case of side injection. In still a further embodiment the snake electrodes are extended in a straight line into the monitoring chamber 152. This will result in the cells being held between the electrodes in the electrical field cage until they are situated in the correct position (a straight line) within the monitoring chamber 152.

When electrodes are placed on both top and bottom substrates 164, 165, then the voltage to all electrodes 160-163 can be switched off simultaneously. Alternatively, however, the electrodes 160-163 can be split into two groups and switched off sequentially. Sequentially is preferred with the bottom electrodes 162, 163 being switched off first and then at a later period the top electrodes 160, 161. The reason for this is that the resulting asymmetric field will effectively push the cells towards the low field region at the bottom of the chamber 153 where they can adhere. As has been found this reduces the time required for adherence and thus also the total assay time. Alternatively, the cells could be confined to a well-defined starting position by using two parallel in-plane electrodes, similar to the barrier electrodes shown in Fig. 17.

According to another embodiment the cells again enter via the buffer inlet but rather than putting electrical field barriers in the branch structure (as shown in Fig. 17), the electrical field barriers are placed in the monitoring chamber 172 as shown in Fig. 19. Since there are no barriers in the tree structure the cells will emerge into the monitoring chamber 172 through all channels of the branch structure (not just the one with only buffer) and will be spread over the monitoring chamber 172 in the direction of the gradient. This has the advantage of decreasing the speed needed to load a given number of cells in the monitoring chamber 172, but is of course not a good start point for the chemotaxis assay. However, by arranging slanted electrodes 173 within the monitoring chamber 172 as shown in Fig. 19, the cells (irrespective of where they enter) are funneled to the (left) side with only buffer (left side in Fig. 19) where they form a straight line. Preferably, an additional vertical electrode 174 is provided on this side to support this process. In the embodiments explained above with reference to Figs. 17 to 19 cells have been introduced via the buffer inlet, whereas in the embodiments described before a side inlet into the monitoring chamber is usually used. If for some reason this configuration is still preferred then it is of course possible to provide such a side inlet and still use a simple electrical field barrier 183, e.g. comprising two (or more, e.g. four, parallel electrodes), in the monitoring chamber 182 to define a start position as shown in Fig. 20.

The embodiments explained above with reference to Figs. 18 to 20 suggest placing electrodes in the monitoring chamber to align the cells through dielectrophoresis (DEP). This principle is applicable to a wide range of chemotaxis devices. In addition to the electrode configurations explained above other electrode arrangements are possible. It is specifically not limited to the tree structure.

An embodiment, in which the cells are introduced into the monitoring chamber 192 from an opening 190 situated in the pure buffer branch 193 of the branch structure 191 is shown in Fig. 21, where this inlet 190 is positioned in the left-most branch 193 just above the monitoring chamber 192. As with previous embodiments, the advantage thereof is that the cells are introduced in the direction of laminar flow and so the start position will be better defined. This embodiment can obviously also contain elements of the other embodiments, e.g. electrodes which are situated in the monitoring chamber 192.

The electrodes in the above shown electrically assisted assays can either be driven directly by separate voltage sources or, alternatively, the electrodes can be driven via large area integrated electronics such as LTPS. If such a platform is used then it is possible to integrate other components into the device such as heaters, temperature sensors, pumps, valves, or sensing electrodes to measure the position of the cells. For example, an integrated heater and temperature sensor could be incorporated into the monitoring chamber to maintain a stable environment for the cells. Alternatively, pumps/valves could be electrically controlled or even the position of cells sensed via electrical methods as described above.

While the present invention has been described in particular detail, it should also be appreciated that numerous modifications are possible within the intended spirit and scope of the invention. Neither is the invention limited to a particular number or type of sensors, nor to a particular type of stimulant gradient chamber. The elements and features of the above described embodiments do not only work in the shown combinations, but can also be combined differently. Generally, all elements can be arbitrarily combined. In interpreting the appended claims it should be understood that: a) the word "comprising" does not exclude the presence of elements other than those listed in a claim; b) the word "consisting" excludes the presence of elements other than those listed in a claim; c) the word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements, d) any reference signs in the claims do not limit their scope; and e) several "means" may be represented by the same item of hardware or software implemented structure or function.

Claims

CLAIMS:

1. Device for the monitoring of the movement of cells comprising: a stimulant gradient chamber (1) for providing a concentration gradient of a stimulant, said stimulant gradient chamber (1) having a cell inlet (12) for the inlet of cells

(C), - an electrical sensor (2) placed in a measurement area for measuring an electrical parameter which changes in response to a movement of cells (C) in the surrounding of said sensor (2) and for generating a detection signal, and a signal evaluation unit (3) for evaluating said detection signal.

2. Device as claimed in claim 1, wherein said sensor (2) is adapted for measuring a capacitance, resistivity and/or conductivity, in particular by measuring a current, a voltage and/or permittivity.

3. Device as claimed in claim 1, wherein said sensor (2 A, 2B) comprises at least two electrodes (20, 21) located on the same substrate (22) or opposite to each other on opposite substrates (22, 23).

4. Device as claimed in claim 1, wherein said sensor (2) comprises a plurality of electrodes (20, 21) arranged in the form of a matrix.

5. Device as claimed in claim 4, wherein said plurality of electrodes (20, 21) comprise a common reference electrode (21), the spacing between said common reference electrode (21) and the other electrodes (20) being provided for being filled with cells and concentration gradient of stimulant.

6. Device as claimed in claim 4, wherein said plurality of electrodes (20, 21) are connected to said evaluation unit (3) via switching elements (26), in particular active electronic switching elements, for individually measuring the detection signals of the individual electrodes.

7. Device as claimed in claim 1, wherein said sensor (2C) comprises a transistor or a metal-insulator-semiconductor diode and adhesion promoting molecules (31) arranged on the gate electrode (30) of said transistor or an electrode of said diode, respectively, for capturing cells (C), indicated by a change of one or more electrical parameters of said transistor or diode, respectively.

8. Device as claimed in claim 3, wherein the spacing between the electrodes (20, 21) and/or the width of the electrodes is adapted such that a cells fills a substantial portion, in particular at least 30%, preferably at least 50%, of the volume between the electrodes.

9. Device as claimed in claim 3, wherein said substrate (22, 23) is made of a material having a low permittivity, in particular a material having a permittivity below 20, preferably below 10, for instance a plastic material.

10. Device as claimed in claim 1, further comprising a gas inlet (13) for the inlet of gas, air and/or liquid having a low electrical permittivity, in particular of gas or air bubbles or oil, into the measurement area (102) prior to measurement.

11. Device as claimed in claim 1 , wherein said stimulant gradient chamber (100) includes a branch structure (101) having a stimulant inlet (10) for the inlet of a stimulant, a buffer inlet (11) for the inlet of a buffer solution, a plurality of interconnected branches for mixing said stimulant and said buffer solution into said concentration gradient of said stimulant, and a concentration gradient outlet (103) for the outlet of said concentration gradient, and a monitoring chamber (102) having a concentration gradient inlet (104) connected to said concentration gradient outlet (103) for the inlet of said concentration gradient and housing said sensor (2).

12. Device as claimed in claim 11, wherein said cell inlet (12) coincides with or is arranged close to said buffer inlet (11) and wherein at junctions of said branch structure (141) barrier electrodes (143) are arranged, each comprising at least two barrier electrode elements (143) being driven with drive signals having a phase lag for preventing the passing of cells through said electrodes.

13. Device as claimed in claim 11, wherein said cell inlet coincides with or is arranged close to said buffer inlet (11) and wherein snake electrodes (160-163) are arranged at the inner walls of branches (153) of said branch structure (151), said snake electrodes being split into at least two snake electrode elements, each being arranged along the direction the branches and being driven with drive signals having a phase lag for avoiding the adherence of cells to the inner walls of the branches.

14. Device as claimed in claim 13, wherein said snake electrodes (160-163) are prolonged into said monitoring chamber (152).

15. Device as claimed in claim 11, wherein said cell inlet (190) is arranged close to the monitoring chamber (192) in a branch of said branch structure (191) leading into said monitoring chamber (192) close to one of its edges.

16. Device as claimed in claim 1, wherein said stimulant gradient chamber (110) includes a stimulant chamber (111) having a stimulant inlet (10) for the inlet of a stimulant, a buffer chamber (112) having a buffer inlet (11) for the inlet of a buffer solution, a plurality of channels (113) connecting said stimulant chamber (111) and said buffer chamber (112), a tapping means (114) for tapping said channels (113) at different tapping locations (115), the distance of said tapping locations (115) from said stimulant chamber (111) being gradually increasing from one channel to the next channel, said tapping means (114) being provided for creating said concentration gradient by tapping at said different tapping locations (113), a concentration gradient outlet (116) for the outlet of said concentration gradient, and a monitoring chamber (117) having said cell inlet (12) and a concentration gradient inlet (118) for the inlet of said concentration gradient and housing said sensor (2).

17. Device as claimed in claim 11 or 16, wherein in said monitoring chamber (172; 182) barrier electrodes (173; 183) are arranged, each comprising at least two barrier electrode elements being driven with drive signals having a phase lag for preventing the passing of cells through said barrier electrodes.

18. Device as claimed in claim 1, wherein said stimulant gradient chamber (130) includes a plurality of parallel micro fluidic channels (131, 132) located parallel to said sensor (2), each second channel (131) being provided for the inlet of said stimulant and the other channels (132) being provided for the inlet of a buffer solution, wherein the walls (133) of said channels (131, 132) are porous to said stimulant and said buffer solution to allow said stimulant and said buffer solution to diffuse through said walls for creating said concentration gradient between neighbor ring channels (131, 132).

19. Device as claimed in claim 18, wherein said walls (133) are made of a gel type material, in particular a hydrogel.

20. Device as claimed in claim 18, wherein said stimulant gradient chamber (130) further includes porosity control means (134, 135) for controlling the porosity of the walls (133) of said channels (131, 132).

21. Device as claimed in claim 20, wherein said porosity control means includes porosity control electrodes (135) arranged close to said walls (133) and wherein said walls (133) are made responsive to an electric voltage applied to said porosity control electrodes or an associated pH change.

22. Device as claimed in claim 1, wherein said stimulant gradient chamber (120) includes a stimulant chamber (121) having a stimulant inlet (10) for the inlet of a stimulant, - a buffer chamber (122) having a buffer inlet (11) for the inlet of a buffer solution, a diffusion chamber (123) connecting said stimulant chamber (121) and said buffer chamber (122) for creating said concentration gradient by diffusion of said stimulant and said buffer solution.

23. Method for the monitoring of the movement of cells (C) comprising the steps of: providing a concentration gradient of a stimulant in a stimulant gradient chamber (1) having a cell inlet (12) for the inlet of cells (C), - measuring an electrical parameter which changes in response to a movement of cells (C) in the surrounding of an electrical sensor (2) placed in a measurement area, generating a detection signal from the measured electrical parameter, and evaluating said detection signal.