WO2008117192A1

WO2008117192A1 - A chemotaxis assay based on chemotactic agents releasing carrier structures

Info

Publication number: WO2008117192A1
Application number: PCT/IB2008/050950
Authority: WO
Inventors: Dennis Merkle; Marcel R. Bohmer; Thomas J. De Hoog; Murray F. Gillies; Simone I. E. Vulto
Original assignee: Koninklijke Philips Electronics N. V.
Priority date: 2007-03-26
Filing date: 2008-03-14
Publication date: 2008-10-02

Abstract

The present invention is concerned inter alia with a method of detecting movement of cells by dielectrophoresis. The present invention is further concerned with providing a dielectrophoresis device for detecting movement of cells.

Description

A CHEMOTAXIS ASSAY BASED ON CHEMOTACTIC AGENTS RELEASING CARRIER STRUCTURES

SUBJECT OF THE INVENTION

The present invention is concerned with a method of detecting movement of cells. The present invention is further concerned with providing a device for detecting movement of cells. BACKGROUND OF THE INVENTION

Cell migration is a key physiological process among prokaryotes as well as eukaryotes.

For eukaryotes it has been shown that impaired cell migration can be involved in pathological processes. In mammalians and particularly humans, typically only stem cells, leukocytes, fibroblasts, tumour cells and sperm cells are capable of active and autonomous migration. For stem cells, migratory capacity is essential in order to allow effective formation as well as regeneration of specific tissues and organs at various parts of the human or animal body. Similarly, for leukocytes it is essential to be able to move throughout the body in order to maintain their immunological guard function and to respond to pathogenic insults.

Movement of cells through e.g. the human body is a complex cellular process that is mediated by a plethora of signals including ligands and receptors such as e.g. the integrin receptor family. Growth factors are one type of molecule which are frequently involved in cellular movement at least in case of mammalians. The modulation of growth factor related signals thus represents a promising target mechanism for various disease treatments and is especially promising for cardiac and vascular diseases (see e.g. Waltenberger et al, (2000) Circulation, 102.2:185-195).

In view of the fundamental physiological importance of cell movement, different assays have been developed in order to investigate cell migration in an in vitro setting, e.g. outside the human or animal body. Typically such assays are classified as two-dimensional migration assays or three-dimensional migration assays.

One of the most common two-dimensional chemotaxis migration assays makes use of the so-called modified Boyden chamber assay.

Typical three-dimensional migration assays include the "under-agarose assay", the "Dunn chamber assay" and the "3D chemotaxis chamber for stabilized gradients" assay.

All of these assays have certain advantages and disadvantages and have in common that it usually takes a rather elongated time period in order to assess the chemotactic behavior of a certain cell type. An overview on various chemotaxis assays can be found in Entschladen et al. (Exp. Cell Res., (2005), 307.2:418-426).

Nevertheless, there is a continuing need in the art for further chemotaxis assays that allow efficient and straight forward analysis of a cell's capacity to move and to react towards certain migratory stimuli.

OBJECT AND SUMMARY OF THE INVENTION

It is one objective of the present invention to provide a method, which allows detecting the migratory behavior of cells in a convenient and efficient setup.

It is a further objective of the present invention to provide devices that allow performing such methods

These objectives as well as others which will become apparent from the ensuing description are attained by the subject matter of the independent claims. Some of the more specific embodiments of the present invention are defined by the dependent claims.

The present invention in one embodiment relates to a method of detecting movement of at least one cell comprising the steps of: providing at least one carrier structure comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; monitoring movement of said at least one cell to or from said at least one carrier structure.

The present invention in another embodiment relates to a method of detecting movement of at least one cell comprising the steps of: - providing at least one carrier structure comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; providing a device which allows to position said at least one carrier structure and said at least one cell to different parts of the device to create a distance between said at least one carrier structure and said at least one cell that allows monitoring movement of said at least one cell to said at least one carrier structure; monitoring movement of said at least one cell to said at least one carrier structure. The present invention in one of the preferred embodiments relates to a method of detecting movement of at least one cell comprising the steps of providing at least one polarizable microcarrier bead comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; - providing a device with at least one first and at least one second electrode; performing dielectrophoresis to localize said at least one microcarrier bead to one of the electrodes; monitoring movement of said at least one cell to or from said at least one microcarrier bead which is localized at said electrode.

The present invention in one of the other preferred embodiment also relates to a method of detecting movement of at least one cell comprising the steps of providing at least one polarizable microcarrier bead comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; providing a device with at least one first and at least one second electrode wherein said first and second electrode are distanced from each other to form an arrangement with a distance between said first and second electrode that allows monitoring movement of said at least one cell from said second electrode to said first electrode; performing dielectrophoresis to localize said at least one microcarrier bead to said first electrode and said at least one cell to said at least second electrode; monitoring movement of said at least one cell from said second electrode to said at least one microcarrier bead which is localized at said first electrode.

The present invention in another preferred embodiment relates to a device for detecting movement of at least one cell comprising: at least one first electrode being capable of immobilizing at least one polarizable microcarrier bead thereto by way of dielectrophoresis; at least one second electrode being capable of immobilizing at least one cell thereto by way of dielectrophoresis; wherein said first and second electrode are distanced from each other to form an arrangement with a distance between said first and second electrode that allows monitoring movement of said at least one cell from said second electrode to said first electrode.

Typically said microcarrier beads have a diameter of about 20 μm to about 1000 μm. A size of about 75 μm to about 350 μm, of about 100 μm to about 200 μm and about 150 μm can be preferred. Said microcarrier beads are made from a material that is polarizable and thus can experience a force in an inhomogeneous electrical field. The beads can thus be moved by dielectrophoresis. In general, said microcarrier beads will comprise bead materials selected from the group comprising polystyrene and poly-lactide.

The microcarrier beads are modified to comprise and to be capable of releasing at least one chemotactic agent.

Such chemotactic agents can be chemoattractants and/or chemorepellants. Chemoattractants will be typically selected from the group of growth factors, hormones, peptides, cytokines, chemokines, cell adhesion molecules (CAMs), integrins, drugs and vitamins.

The chemotactic agents may be contacted with the microcarrier bead in the form of a (isolated) molecule or they may themselves be released from e.g. a cell that adheres to said microcarrier bead.

The cells which can be analyzed by the method in accordance with the present invention as to their movement capacity, are selected from prokaryotic or eukaryotic cells. Among prokaryotic cells, cells of e.g. the genus Enterobacteria can be analyzed. A typical representative is Escherichia coli. Prokaryotic pathogens such as Mycobacterium tuberculosis or Helicobacter pylori will also be interesting cells to study.

Within eukaryotic cells, particularly cells of vertebrates and more preferably of mammalians can be analyzed. Such cells include e.g. stem cells, leukocytes, fibroblasts, tumor cells, sperm cells, fetal cells etc. In a preferred embodiment the cell types may be of human origin.

The methods in accordance with the present invention may be performed as a two-dimensional or three-dimensional chemotactic assay. For a two-dimensional assay, a device will be selected that allows to locate carrier structures and cells to different parts of the device to create a distance between the carrier structures and the cells allowing to monitor movement of the cells to the carrier structure.

A device as it is used for dielectrophoresis will typically comprise at least two electrodes which are positioned in distance from each other to create e.g. a channel- like structure that is sufficiently dimensioned to allow monitoring translocation of a cell from one electrode to the other.

However, in another embodiment, the invention may allow the analysis of the three-dimensional movement behaviour of a cell. To this end, one may position a three-dimensional porous structure between the carrier structures and the cells. If a device for dielectrophoresis is used, the structure will e.g. be positioned between the aforementioned electrodes. Alternatively and/or additionally one can position such porous three-dimensional structures on e.g. the carrier structures such as the afore- described microcarrier beads.

These structures will constitute an additional barrier that allows analyzing the capacity of a cell to respond to and circumvent and/or penetrate a movement barrier.

Furthermore, the methods in accordance with the present invention may be performed under flow control which means that the device in which the method is performed is designed to allow for constant replenishing of liquids such as buffers in proximity of the cells that are localised to one part of the devices such as at one of the afore described electrodes if dielectrophoresis is used to locate cells and carrier structures.

The present invention also relates to the use of carrier structures and preferably of microcarrier beads which comprise and are capable of releasing a chemotactic for determining the migratory behaviour of cells. The present invention further relates to the use of the aforementioned devices which can be used in dielectrophoresis for determining the migratory behaviour of cells.

FIGURE LEGENDS

Fig. 1 schematically depicts the principle of the present chemotaxis assay for one embodiment. Microcarrier beads which release a chemoattractant are localised to a first electrode (pair) by dielectrophoresis while cells are localised to a second electrode (pair) by dielectrophoresis. The release of the chemoattractant creates a gradient along which the cells migrate. Fig. 2schematically depicts a further embodiment of the present invention. Again microcarrier beads which comprise a chemoattractant are positioned at a first electrode (pair) by dielectrophoresis while a certain type of cell such as e.g. leukocytes are positioned at a second electrode (pair) by dielectrophoresis. The leukocytes have themselves been isolated by use of microcarrier beads being functionalized with a cell- specific factor (e.g. VCAM-I).

Fig. 3 schematically depicts a two-dimensional and three-

5 dimensional setup of the method in accordance with the present invention. In Fig. 3a, microcarrier beads are positioned at a first electrode (pair) by dielectrophoresis while cells are positioned at a second electrode (pair) by dielectrophoresis. The microcarriers release a chemoattractant thereby creating a gradient along

10 which the cells can travel. In Fig. 3b, a three-dimensional porous structure (e.g. a collagen or fibronectin matrix) is positioned between a first and second electrode (pair) which allows to analyze the movement of the cells along the gradient in a three- dimensional setup.

15 Fig. 4shows further embodiments of the chemotaxis assay in accordance with the present invention. Fig. 4a depicts a microcarrier bead which is covered with a three-dimensional matrix. In Fig. 4b, a microcarrier bead is used which is covered with a cell layer. The cell layer of the microcarrier bead can

20 release a chemoattractant which then forms a gradient and allows monitoring movement of cells from a second electrode (pair) to which the cells have been trapped via dielectrophoresis. Fig. 5 shows a further embodiment of the present invention in which flow control is incorporated into the device which is used

25 to perform the chemotaxis assay of the present invention. Thus, microcarrier beads which are capable of releasing a chemoattractant are positioned at a first electrode (pair) by dielectrophoresis while cells are positioned at a second electrode (pair) by dielectrophoresis. The release of the chemoattractant

30 creates a gradient. In order to avoid that diffusion overtakes the formation of the gradient a flow control can be applied near the cell surfaces in order to replenish fluids such as buffer.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the finding that one can use carrier structures which comprise and are capable of releasing a chemotactic agent to monitor the migratory behaviour of cells. To this end, carrier structures are localised to one part of a device while cells are localised to another part of the device to create a distance between the carrier structures and the cells that is of sufficient dimensions to allow monitoring of cell migration. By release of the chemotactic agent which may be e.g. a chemoattractant, a signal gradient will develop along which the cells may move if they display affinity towards the chemoattractant.

Before some of the embodiments of the present invention are described in more detail, the following definitions are introduced. As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise. Thus, the term "a microcarrier bead" can include more than one microcarrier bead, namely two, three, four, five etc. microcarrier beads.

The terms "about" and "approximately" in the context of the present invention denotes an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of +/- 10%, and preferably +1- 5%.

As set out above, the invention relates to a method of detecting movement of at least one cell comprising the steps of: - providing at least one carrier structure comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; monitoring movement of said at least one cell to or from said at least one carrier structure. As set out above, the invention also relates to a method of detecting movement of at least one cell comprising the steps of: providing at least one carrier structure comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; providing a device which allows to locate said at least one carrier structure and said at least one cell to different parts of the device to create a distance between said at least one carrier structure and said at least one cell that allows monitoring movement of said at least one cell to said at least one carrier structure; monitoring movement of said at least one cell to said at least one carrier structure.

The term "carrier structure" refers to any three dimensional preferably solid structure which comprises and is capable of releasing a chemotactic agent. The person skilled in the art will be aware that the carrier structures may have different forms and dimensions. For example, a carrier structure may be substantially round and have the form of a bead. A chemotactic agent may be incorporated and/or contacted with such a structure in different ways. One may e.g. use a matrix-like, porous bead which incorporates a chemotactic agent and releases the same over time. Alternatively and/or additionally one may coat a bead with a chemotactic agent and dispose thereon a film coating that ensures release of the chemotactic agent over time. While one specific embodiment of the present invention uses microcarrier beads as carrier structures and dielectrophoresis to locate carrier structures and cells to different parts of the above-mentioned devices, the present invention is thus concerned with the use of carrier structures and preferably microcarrier beads which comprise and are capable of releasing a chemotactic agent for analyzing the migratory behaviour of cells.

In one embodiment the present invention uses the above described general findings by relying on polarizable microcarrier beads which comprise and are capable of releasing a chemotactic agent to study the movement of cells. Due to the different polarization characteristics, microcarrier beads and cells will experience different forces in an inhomogeneous electrical field and can thus be separated by dielectrophoresis efficiently to e.g. separate electrodes. If the microcarrier beads are designed to release a chemoattractant this will create a signal gradient of chemoattractant between the microcarrier beads and the cells which are localized to different parts of the dielectrophoresis device. Once cells start sensing the chemoattractant, they will start moving towards the microcarrier beads along an increasing concentration of the signal gradient. Thus, the present invention allows studying the capacity of cells to respond to chemotactic agents and to study their behaviour as well as their migratory capacity.

As set out above, the invention in one of the preferred embodiments thus relates to a method of detecting movement of at least one cell comprising the steps of - providing at least one polarizable microcarrier bead comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; providing a device with at least one first and at least one second electrode electrode; - performing dielectrophoresis to localize said at least one microcarrier bead to one of the electrodes; monitoring movement of said at least one cell to said at least one microcarrier bead which is localized at said electrode.

As set out above, the invention in one of the other preferred embodiments relates to a method of detecting movement of at least one cell comprising the steps of providing at least one polarizable microcarrier bead comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; providing a device with at least one first and at least one second electrode wherein said first and second electrode are distanced from each other to form an arrangement with a distance between said first and second electrode that allows monitoring movement of a cell from said second electrode to said first electrode; performing dielectrophoresis to localize said at least one microcarrier bead to said first electrode and said at least one cell to said at least second electrode; monitoring movement of said at least one cell from said second electrode to said at least one microcarrier bead being localized at said first electrode.

The term "microcarrier bead" denotes a polarizable structure which can experience a force in an inhomogeneous electrical field and which has a diameter in the range of approximately 20 μm to approximately 1000 μm. A diameter in the range of approximately 75 μm to approximately 300 μm and more frequently in the range of approximately 100 μm to approximately 200 μm can be preferred.

In some of the preferred embodiments, the beads will display a diameter in the range of about 120 μm to about 180 μm or about 150 μm.

The beads may have any form that is suitable for the purposes of the present invention. They thus may have a rectangular shape, a triangular shape etc. Beads with a round or a substantially round form can be preferred according to the present invention. Thus, the beads will typically have a round, elliptical or spheroid form. There are no specific requirements as to the materials from which the beads are made except that the beads must be polarizable in an inhomogeneous electrical field to experience a force if dielectrophoresis is performed on the beads. A further requirement is that the beads should be designed to comprise and allow release of a chemotactic signal. Typically the beads should be made in such a way that a chemotactic signal is released over a prolonged period of time which means that the beads typically release the chemoattractant over about at least 10 minutes up to about 5 hours or over at least about 30 minutes up to about 4 hours. A release of the chemotactic agent over about 1 hour or about 2 hours may also prove suitable. With respect to the aforementioned requirements, the microcarrier beads may be produced from materials such as polystyrene, polydivnylbenzene and polymethylmethacrylate or e.g. from biodegradable polymers such as poly-lactides, polyclycolides, polycaprolacton and copolymers thereof.

Such beads are in principle commercially available from e.g. SoloHill Engineering, Inc (Ann Arbor, Michigan, USA). The beads may be further modified, e.g. coated in order to ensure adherence of the chemotactic agents or a certain type of cell. Such coatings include glass, collagen (gelatine), fibronectin, a cationic surface treatment, recombinant proteins such as growth factors and cell adhesion molecules including vascular cell adhesion molecule 1 (VCAM-I), antibodies etc.

In the context of the present invention the term "chemotactic agent" denotes chemoattractants as well as chemorepellants. Chemoattractants are understood to stimulate migration of a cell towards this agent while chemorepellants are understood to stimulate the opposite behaviour. In the context of the present invention the term "chemoattractant" typically denotes chemical substances as well as peptides and proteins as they are known to induce migration of cells. Such chemoattractants typically include growth factors, hormones, peptides, cytokines, chemokines, cell adhesion molecules (CAMs), integrins, drugs and vitamins. Chemoattractants such as growth factors, hormones, cytokines, chemokines can be preferred as they are known to attract and induce a migratory behaviour in eukaryotic cells such as human or animal mammalian cells.

Typical growth factors include e.g. human growth factor, human nerve growth factor, platelet derived growth factor, TGFB, EGF, VEGF, TNF-a,TNF-b, FGFs, TGF-a, EPO, IGF-I, IGF-II, IL-I, IL-2, IL-6, IL-8, INF-g and CSFs as well as integrins and CAMs including VLA-I to VLA-6, LPAM-I, LFA-I, CR3, CR4, leukointegrin, collagen, laminin, fibronectin, VCAM-I, MAdCAM-I, E-cadherin, ICAM-I, ICAM-2, ICAM-3, C3b, C4b.

Chemotactic agents for the purposes of the present invention, however, do not only relate to single molecules, complexes of molecules or mixtures of molecules that can stimulate a migratory behaviour in a cell but also relate to cells which themselves e.g. secrete migratory stimuli for other cells. Thus, a microcarrier bead in accordance with the present invention can comprise e.g. on its surface cells that segregate a chemokine which induces a migratory or adhesion behaviour in other cells. Such factors include e.g. human growth factor, human nerve growth factor, platelet derived growth factor, TGFB, EGF, VEGF, TNF-a, TNF-b, FGFs, TGF-a, Epo, IGF-I, IGF-II, IL-I, IL-2, IL-6, IL-8, INF-g and CSFs as well as integrins and CAMs including VLA-I to VLA-6, LPAM-I, LFA-I, CR3, CR4, leukointegrin, collagen, laminin, fibronectin, VCAM-I, MAdCAM-I, E-cadherin, ICAM-I, ICAM-2, ICAM-3, C3b, C4b, C5a. The person skilled in the art is familiar with the production of microcarrier beads. Thus, one may obtain these beads from aforementioned commercial sources or produce them according to the protocols being described in e.g. Gu et al (J. Control Release (2004), 96.3:463-472), Li et al (Acta Pharmacol. Sin. (2006) 27.6:754- 759), Norrgren et al (Dev. Biol. Stand. (1983), 55:43-51) or Tatard et al (Biomaterials (2005), 26.17:3727-3737).

Microcarrier beads can e.g. be synthesized e.g. by controlled emulsifϊcation techniques such as submerged ink-jet printing.

If a microcarrier bead is to be produced from a polymer such as polystyrene, a polymer solution in a solvent such as dichloromethane can be ink-jetted into an aqueous phase containing a stabilizer for the monodisperse emulsion that is formed. To this end, one can use e.g. polyvinyl alcohol but also proteins could be used to achieve efficient stabilization. Subsequently polymer beads with a narrow size distribution are formed upon removal of the solvent.

If one intends to incorporate chemoattractants such as a growth factors which can be water soluble, a double emulsion technique can be used. To this end, the water soluble chemoattractant may be first dissolved in an aqueous phase and this aqueous phase is emulsified in the polymer solution. If necessary or desired, additional stabilizers such as e.g. Pluronic can be added. This first emulsion is then ink-jetted into a second aqueous phase and beads are again formed upon removal of the solvent. The person skilled in the art will be aware that microcarrier beads of different properties can be produced. Thus, for example microcarrier beads may be produced which incorporate a chemoattractant such as a growth factor such that release of the chemoattractant from the microcarrier bead is mainly governed by diffusion. Such a microcarrier bead may comprise the chemoattractant in a diffusion matrix. If on the other side the microcarrier bead is produced from a biodegradable and/or swellable substance, the release of the chemoattractant may be governed by diffusion as well as erosion principles.

The person skilled in the art is aware how chemotactic agents such as a protein growth factor, a small molecule etc. may be e.g. incorporated into a microcarrier bead structure in order to render the microcarrier bead capable of releasing the chemoattractant over time. If a microcarrier bead is e.g. produced from poly-lactide which is a bio-degradable polymer together with a chemoattractant, the microcarrier will start degrading upon contact with a fluid and thereby release the chemoattractant.

However, the person skilled in the art is also well familiar on how to grow cells on a microcarrier bead if cells are used in order to release the chemotactic agent. Thus, one may for example add microcarrier beads such as those available from SoloHill (vide supra) to a cell culture of e.g. mammalian cells. If e.g. a suspension cell culture is gently stirred under appropriate conditions, the cells will start adhering to the microcarriers and will form layers of cells on microcarrier beads. The cell-coated microcarrier beads may then be used in the methods in accordance with the invention.

In the context of the present invention the term "cell" denotes cells, the migratory behaviour of which is to be examined. Thus, the term may relate to any type of cell that can be isolated from a biological sample such as bodily fluids including blood, plasma, urine etc. However, samples may also comprise environmental samples taking from soils, lakes, rivers, plants etc.

Typically one will use a sample for which one suspects that it comprises cell types that are known to have a potential for migratory capacity. In case of mammalian and particularly human cells such cell types include e.g. stem cells, leukocytes, fibroblasts, tumour cells, sperm cells and fetal cells. The dielectrophoresis devices which are to be used in the methods in accordance with the present invention comprise: at least one first electrode being capable of immobilizing at least one polarizable microcarrier bead thereto by way of dielectrophoresis; and at least one second electrode being capable of immobilizing at least one cell thereto by way of dielectrophoresis; wherein said first and second electrode are distanced from each other to form an arrangement with a distance between said first and second electrode that allows monitoring movement of a cell from said second electrode to said first electrode.

Such an arrangement of electrodes can take a chamber-like or channel- like appearance.

The person skilled in the art is aware that dielectrophoresis is a method of manipulating and/or moving particles by application of a non-uniform, i.e. an inhomogeneous electrical field. When an electrically uncharged particle such as e.g. a cell is placed in such a field, it will become polarized and it will experience a driving force due to the non-uniformity of the cell. This force can either be in the direction of increasing field strength (so-called positive dielectrophoresis) or decreasing field strength (negative dielectrophoresis). As the person skilled in the art is aware, the frequency required for dielectrophoresis can be dependent on the size and the composition of particles such as microcarrier beads, cells etc. The person skilled in the art is furthermore aware that usually the requirements for using dilectrophoresis to move a microcarrier bead or a cell in an inhomogeneous electrical field will differ considerably so that these differences and properties can be used to localize microcarrier beads as well as cells to different parts of a dielectrophoretic device.

Thus, dielectrophoresis will typically occur when at least two electrodes are utilized to create at least two regions of electric field within a device, with the first region having a high field and the second region having a low field. Based on the size and surface characteristics of the beads/cells they will either be attracted to the high field region (positive dielectrophoresis) or be repelled into a low field region (negative dielectrophoresis) . The person skilled in the art will be aware that the devices and methods in accordance with the invention may use more than two electrodes such as for example at least two pairs of electrodes as this will allow to achieve the different field strengths more easily. Thus where the figures denote an "electrode", this may mean an electrode pair to create the different field strengths at the desired parts of the device. However, the device may also comprise quadrupole electrodes etc. The dielectrophoretic device that can be used for separating microcarrier beads and cells in accordance with the methods of the present invention comprise the elements as described above. Typically, these devices will comprise at least two electrodes and preferably at least two electrode pairs being distanced from each other to form a channel- like structure such as depicted in Figs. 1 to 5.

The electrodes typically comprise any conductive material, that can function as an electrode and is, preferably, biologically inert and biologically compatible. It may be transparent. Typically metals include platinum, titanium, gold, tantalum and ITO. This material can also contain thin insulting layers (including hydrogels). In a typical embodiment the electrodes will take the form of a thin metal form being formed from the aforementioned metals on a support such as a glass substrate.

The size of the channel- like structures which are created by e.g. electrodes should be such that cells and carrier structures such as microcarrier beads can be accommodated on different sides of the channel with a distance in between them that is of sufficient dimension to allow a meaningful cell migration distance. Clusters of cells will typically have a diameter of about 10 to about 100 μm. Microcarrier beads in accordance with the invention will typically have a diameter of about e.g. 75 to about 350 μm and a typical meaningful cell migration distance will be in the range of about 10 to about 1000 μm, preferably around 10-50 μm. Therefore, the distance between the electrodes and the dimension of the channel- like structures should be in the order of about 200 μm to about 800 μm. Another useful distance lies in the range of approximately 300 μm to approximately 600 μm or around about 400 μm.

The principle outlay of the methods in accordance with the invention is schematically depicted in Fig. 1. As has been mentioned above, dielectrophoresis uses the different polarization characteristics of particles to separate them in an inhomogeneous electrical field. As the microcarrier beads of the invention and cells differ significantly as to their size and composition they have different polarization characteristics and one can use e.g. positive dielectrophoresis to separate these two components in a typical dielectrophoresis device. Thus, one can trap e.g. chemoattractant-releasing microcarrier beads to one electrode (pair) while cells are trapped to a second electrode (pair). In Fig. 1 it is depicted that microcarrier beads which comprise a chemoattractant such as a growth factor and cells are added to dielectrophoretic device which is formed by two different electrodes (or electrode pairs). Applying positive dielectrophoresis (pDEP) the chemoattractant-releasing microcarrier beads are trapped to a first electrode (pair) while the cells are trapped to a second electrode (pair). The distance of the electrodes chosen is such that a channel-like structure is created between the two electrodes with a dimension that is useful for observing a migratory behaviour of cells. As the microcarrier beads release the chemoattractant over time a gradient is formed between the electrode (pair) to which the microcarrier beads are trapped, and the electrode (pair) to which the cells are trapped. Once the cells start sensing the chemoattractant they can move along the gradient of the chemoattractant towards the microcarrier beads.

This migratory behaviour of the cells can then be examined. To this end, the device may be a connected e.g. to optical device such as time-lapse video microscopy to allow monitoring of the cells' movement. Furthermore, the cells that have moved towards the microcarrier beads can subsequently be quantified and the migratory capacity of the cells which have been originally introduced in the dielectrophoretic device can be calculated.

Thus, the methods (and devices) in accordance with the invention allow for a quantitative as well as qualitative determination of the migratory capacity of cells. A further embodiment of the present invention is schematically depicted in Fig. 2. This figure describes a combination of pre-selecting a certain type of cells and subsequent studying of their migratory capacity. To this end one may use two types of microcarrier beads. The first type of microcarrier beads comprises e.g. a chemoattractant such as a growth factor. The second type of microcarrier beads is functionalized on its surface with a probe molecule that is specific for a certain cell type. Thus, one may e.g. use microcarrier beads which have been amended to display a cell adhesion matrix protein such as VCAM-I. One can use such probe-modified microcarrier beads to select for certain types of cell within a cellular sample. If one then inserts the microcarrier beads comprising a chemoattractant and the probe-modified microcarrier beads which have been covered with the certain type of cells within a dielectrophoretic device, one can localize these two types of microcarrier beads depending on their properties to different electrodes (pairs of electrodes). Again, the microcarrier beads comprising the chemoattractant will release the chemoattractant over time and thereby create a signal gradient along which the cells that have been adhered to the second type of microcarrier beads can migrate. This type of setup allows to e.g. compare the affinity of a certain type of cell for a specific probe molecule such as an extracellular matrix binding protein versus a chemoattractant factor such as a growth factor.

The present invention thus in one embodiment relates to a method detecting the movement of cells in which a polarizable microcarrier bead comprising and capable of releasing a chemotactic agent is localized to one electrode by dielectrophoresis while a second type of polarizable microcarrier beads which are capable of specifically attaching to a certain type of cells are trapped to a second electrode by dielectrophoresis. The two electrodes are distanced from each other and form a channel- like structure of a dimension that is sufficiently large to allow a meaningful determination of cell migration. Upon development of a signal gradient, the cells being specifically attached to the second type of microcarrier beads travel along an increasing signal gradient towards the microcarrier beads which release the chemotactic agent depending on the chemotactic agent's activity. The methods and devices described herein can be easily adapted to allow for determination of different aspects of the migratory capacity of a cell.

Thus, the methods and devices in accordance with the invention can be used to analyze migratory cells within a two-dimensional or three-dimensional environment. The analysis of migratory cells within a two-dimensional setup has been described above in connection with Figs. 1 and 2.

However, by positioning a three-dimensional porous structure between or on the electrodes of the devices described above, it is also possible to determine the migratory behaviour of cells within a three-dimensional environment.

For the purposes of the present invention a "three-dimensional porous structure" refers to a structure with spacings that are sufficiently large to allow penetration of cells there through. Such three-dimensional porous structures may thus have a sponge-like appearance and may be made from e.g. a collagen or fϊbronectin matrix. Other three-dimensional structures that can be used in accordance with the present invention may be a filter, a filter matrix or a hydrogel such as acrylamide and agarose gels. Thus, a three-dimensional porous structure in the context of the present invention has a scaffold-like appearance that allows to determine how migratory cells that react to a chemoattractant stimulus can cope with barrier-like structures on their migration pathway.

If one e.g. compares Fig. 3a and Fig. 3b two different scenarios can be distinguished. In both cases time-controlled release of a chemoattractant will create a signal gradient across the channel-like arrangement being formed by the two electrodes which is sensed by the cells. This gradient thus provides a migratory stimulus for the cells to follow. The cells may then either crawl along the surface of the channel- like arrangement towards the microcarriers in a two-dimensional type assay as depicted in Fig. 3a or through a three-dimensional (matrix) structure such as depicted by Fig. 3b. The person skilled in the art is also aware that other embodiments may replace a three-dimensional structure as described above. For example one may implement a third electrode (pair) at or near the centre of the channel- like arrangement. Such an electrode can be used to trap via dielectrophoresis e.g. microcarrier beads which have been covered with a collagen or fϊbronectin matrix. Using this approach also a three-dimensional structure is formed which builds a migratory barrier within the two electrodes that are used to trap the chemoattractant-containing microcarrier beads and the cells for which the microcarrier behaviour is to be analyzed.

There are further elaborations to the aforementioned methods and devices that can be used to determine the migratory behaviour of a cell.

For example instead of using a three-dimensional structure such as a collagen matrix being positioned between the first and the second electrode one can also use microcarrier beads which comprise and are capable of releasing a chemotactic agent such as growth factor and are furthermore surrounded by a three-dimensional porous structure such as e.g. a collagen matrix. Such an embodiment is depicted in Fig. 4. Thus, Fig. 4a shows a microcarrier bead which is surrounded by e.g. a collagen matrix. If such chemoattractant-containing, collagen-surrounded microcarrier beads are trapped to an electrode via dielectrophoresis again a signal gradient will develop. After cells have been trapped to a second electrode (pair) via dielectrophoresis, the cells may start migrating upon encountering the signal stimulus and travel towards the microcarrier beads. Once the cells reach the microcarrier bead they will first contact the collagen matrix and depending on their properties may rest on the outside of the matrix (scenario 1) or will continue to travel through the collagen matrix to the surface of the microcarrier beads (scenario 2). In a further variant of this aspect of the invention only the microcarrier beads with the three-dimensional porous matrix can be located to an electrode by dielectrophoresis and cells which have been positioned in the device can crawl to and through the matrix as the chemotactic agent is released. Similarly one can trap microcarrier beads with a matrix and cells disposed thereon to an electrode and analyse whether the released chemotactic agent acts as a chemoattractant or chemorepellant.

The present invention in one embodiment thus also relates to a device and method in which polarisable microcarrier beads are used which comprise and are capable of releasing a chemotactic agent and are surrounded by a three-dimensional porous structure. Such microcarrier beads are suitable to allow to determine the influence of certain three-dimensional structures on the migratory behaviour of a cell. If for example a collagen matrix exerts an attractant stimulus on cells, the cells will continue to move through the three-dimensional structure towards the microcarrier beads. If however such a three-dimensional structure acts as a repellant the cells will stop on outer shell of the collagen matrix. This latter approach can be further adapted to study the migratory behaviour of cells within another cell layer. For example it has been set out above that the methods and devices in accordance with the present invention may use polarizable microcarrier beads which are covered with cells that themselves release a chemoattractant. If such cell covered-microcarrier beads are trapped to an electrode (pair) via dielectrophoresis and a signal gradient is established, the cells being trapped at the other electrode (pair) can crawl towards cell covered-microcarrier beads being trapped at the first electrode (see Figure 5). Dependent on whether the migratory cells interact with the cell covered microcarrier beads or not, one will observe stopping of the migration of the migratory cells upon contacting the cell covered-microcarrier beads (scenario 1 of Fig. 4) or one will observe intimate contact and penetration of the migratory cells into the cell layer being disposed on the microcarrier beads (scenario 2 of Fig. 4).

Yet another embodiment of the present invention relates to devices and methods that can be put under flow control of the arrangement created by the carrier structure and the cells and preferably by the afore-mentioned electrodes (electrode pairs). In accordance with the present invention this means that the flow of liquids near the cells being trapped via dielectrophoresis to an electrode can be controlled.

The reason for applying a flow control in proximity to the cells being trapped to an electrode via dielectrophoresis is that the formation of the signal gradient by the released chemotactic agent over time is counteracted by diffusion processes. If however liquids such as buffer are replenished in close proximity to the cells being trapped to an electrode via dielectrophoresis, formation of the gradient is kept stable.

Thus, flow control in the context of the present invention means that the liquids surrounding the cells being positioned at an electrode (pair) via dielectrophoresis are constantly replenished in order to reduce breakdown of the chemoattractant gradient being created by the constant release of the chemoattractant from the microcarrier beads to a minimum.

As a consequence, migration of cells from the electrode to which they are trapped via dielectrophoresis to the chemoattractant releasing-microcarrier beads being positioned at another electrode can be allocated more firmly and reliably to the released signal than to general migratory behaviour.

Different embodiments in which a flow control is applied in proximity to cells being positioned at an electrode via dielectrophoresis are depicted in Fig. 5.

As has been mentioned in the introductory section, studies have demonstrated that a variety of diseases can have an impact on cellular migration. Thus it has been demonstrated that monocyte mobility towards growth factors is e.g. affected in diabetes, hypercholesterolemia and hypertension patients versus healthy individuals. Using the devices and methods in accordance with the invention one can thus identify e.g. patients or individuals whose cells show an abnormal migratory behaviour towards certain chemotactic stimuli. Moreover, the devices and methods in accordance with the present invention may also have beneficial applications in basic research and other applications as they allow to e.g. study the migratory behaviour of cells on a rather short time scale. Furthermore, as has been set out for some embodiments of the present invention, the methods and devices of the present invention can be easily adapted to e.g. study the migratory response of cells to stimuli from other cells and to study the propensity of a migratory cell to penetrate through a three- dimensional barrier or through cell layers.

The present invention has been discussed with respect to some exemplary embodiments. However, this is not meant to limit the scope of the invention in any way.

Claims

CLAIMS:

1. Method of detecting movement of at least one cell comprising the steps of: providing at least one carrier structure comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; - monitoring movement of said at least one cell to or from said at least one carrier structure.

2. Method according to claim 1 comprising the steps of: providing at least one carrier structure comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; providing a device which allows to locate said at least one carrier structure and said at least one cell to different parts of the device to create a distance between said at least one carrier structure and said at least one cell that allows monitoring movement of said at least one cell to said at least one carrier structure; monitoring movement of said at least one cell to said at least one carrier structure.

3. Method according to claim 1 or claim 2comprising the steps of providing at least one polarizable microcarrier bead comprising and being capable of releasing at least one chemotactic agent; providing at least one cell; providing a device with at least one first and at least one second electrode; performing dielectrophoresis to localize said at least one microcarrier bead to one of the electrodes; monitoring movement of said at least one cell to or from said at least one microcarrier bead which is localized at said electrode.

4. Method according to claim 3 comprising the steps of: providing at least one microcarrier bead being polarizable which comprises and is capable of releasing at least one chemotactic agent; providing at least one cell; providing a device with at least one first and at least one second electrode wherein said first and second electrode are distanced from each other to form an arrangement with a distance between said first and second electrode that allows monitoring movement of a cell from said second electrode to said first electrode; performing dielectrophoresis to localize said at least one microcarrier bead to said first electrode and said at least one cell to said at least second electrode; monitoring movement of said at least one cell from said second electrode to said at least one microcarrier bead being localized at said first electrode.

5. Method according to claim 3 or claim 4, wherein said microcarrier bead has a diameter of about 20 μm to about 1000 μm.

6. Method according to any of claims 3 to 5, wherein said microcarrier bead comprises bead materials selected from the group comprising polystyrene, polydivnylbenzene and polymethylmethacrylate and biodegradable polymers such as poly-lactides, polyclycolides, polycaprolacton and copolymers thereof.

7. Method according to any of claims 1 to 6, wherein said chemotactic agent is released from at least one second cell which is in contact with said microcarrier bead.

8. Method according to any of claims 1 to 7, wherein a porous three-dimensional structure is positioned on said carrier structure, preferably on said microcarrier bead.

9. Method according to any of claims 1 to 7, wherein a porous three-dimensional structure is positioned between said carrier structure, preferably between said microcarrier bead and said at least one cell.

10. Device for detecting movement of at least one cell comprising: at least one first electrode being capable of immobilizing at least one polarizable microcarrier bead thereto by way of dielectrophoresis; at least one second electrode being capable of immobilizing at least one cell thereto by way of dielectrophoresis; wherein said first and second electrode are distanced from each other to form an arrangement with a distance between said first and second electrode that allows monitoring movement of said cell from said second electrode to said first electrode.

11. Device according to claim 10, wherein a porous three-dimensional structure is positioned between said first and said second electrode.

12. Use of a device according to claim 10 or claim 11 for detecting movement of at least one cell.

13. Use of at least one carrier structure for detecting movement of at least one cell.

14. Use according to claim 13, wherein said at least one carrier structure, comprises and is capable of releasing a chemotactic agent.

15. Use according to claim 13 or 14, wherein a porous three-dimensional structure is positioned on said carrier structure, preferably on said microcarrier bead.