WO2009147486A2

WO2009147486A2 - Micro-fluidic device for measurements of cell migration in response to chemical stimuli

Info

Publication number: WO2009147486A2
Application number: PCT/IB2009/005712
Authority: WO
Inventors: Marco Pizzi; Emilio Hirsch; Carlotta Costa; Elisa Vitiello
Original assignee: Techfab S.R.L.
Priority date: 2008-05-26
Filing date: 2009-05-25
Publication date: 2009-12-10
Also published as: WO2009147486A3; ITTO20080396A1

Abstract

The invention relates to a device for making measurements of the migration of cells in response to exposure to a gradient of concentration of a chemoat tractive substance (chemotaxis). In particular, the invention relates to a device comprising a first site designed to receive a specimen of cells, and at least one second site designed to receive a substance potentially capable of influencing chemotaxis, wherein the first and second sites are fluidically connected together by a containing duct, and are arranged along an axis A directed between the first site and the second site with a non-uniform density, a plurality of obstacle elements that create along the duct a gradient in the free section of passage at the disposal of the cells, which is defined by a corresponding plurality of micro- channels directed parallel to the axis A and having a pre-set transverse dimension d.

Description

MICROFLUIDIC DEVICE FOR MEASURING CELL MIGRATION IN RESPONSE TO CHEMICAL STIMULI

Field of the art The present invention relates to a device for carrying out measurements of cell migration in response to one or more stimuli of a chemical type (chemotaxis) .

State of tlxe art Chemotaxis is the natural phenomenon whereby cells or microorganisms impose on their movements a preferential orientation in response to a chemical gradient detected in their environment. It constitutes one of the basic cell processes, and its origins are to be sought in a biological mechanism, which has a long history and has evolved in unicellular organisms right from the first stages of phylogeny, with the aim of discriminating, in the environment surrounding the cell, the favourable substances from the harmful ones. Throughout the millennia, chemotaxis has hence evolved until it has become a basic process whereby the cells are capable of detecting a gradient of concentration and orienting their movements in the direction of the increasing/decreasing concentration .

In pluricellular organisms, chemotaxis has a critical importance both in the initial stages of the life cycle (for example, the motion of spermatozoa towards the ovule) and in the immediately subsequent stages of development (for example, migration of neurones or lymphocytes) , as well as then in carrying out the normal vital functions. Leukocytes, for example, detect the concentration of the products of bacterial origin to orient themselves towards the site of the infection where their activity is required by the organism.

In addition to the crucial role of chemotaxis in a multiplicity of biological processes, it has been recognized that the basic mechanisms of chemotaxis can be altered, in animals, in the presence of pathologies such as inflammatory- conditions and formation of metastasis. Consequently, an understanding of the molecular mechanisms underlying chemotaxis could have a significant impact on the possibility of identifying new methodologies of treatment for said conditions .

In recent years the study of the mechanisms underlying chemotaxis has been set underway. Basically, the cells detect the chemotactic gradient by means of receptors located in a region corresponding to the cell membrane. Said receptors are proteins, which usually bind in a selective way chemically distinct species, which can range from small organic compounds to functional groups of large dimensions. The formation of bonds between these chemotactic agonists and their receptors triggers a transduction of signals, which, within the cell, leads to the modification of the cell morphology.

In this phase, there exist different molecules, which, operating as signal amplifiers, play a key role in directing remodelling of the cell structure as a function of the gradient of concentration of the agonist. This process of polarization requires the concerted action of contractile proteins that promote extrusion of the cytoplasm in the anterior part and retraction of the cell body in the posterior part of the cell .

Even though different cells migrate principally as a consequence of said mechanism referred to as "ameboid migration" , other cells need to adhere to the extracellular environment and to proteins of the extracellular matrix. The formation of bonds with these substrates and the consequent contraction of the cytoplasm are, for example, essential in the first stages of activation and of subsequent recruitment of the leukocytes outside the blood flow. Since arresting of the chemotaxis can effectively contribute to the treatment of different illnesses, a considerable effort has been made to define modalities for objective measurement of cell migration within a gradient of chemotactic agonists. Even though it has always appeared desirable to characterize new pharmaceutical compounds by high- throughput screening on the basis of the evaluation of cell migration, the existence of numerous technical difficulties has never enabled effective implementation of an approach of this sort.

Various techniques are known and in use for study of the effect on the cell motility of chemoattractive molecules. Some of them are qualitative and enable determination of whether the cells prefer or otherwise the substance under examination; others are quantitative and supply information on the intensity of the responses in a more detailed way.

Amongst said techniques, the most widespread are: - Agar dish (with single chamber) - the device is obtained by cutting, within semisolid layers of agar agar or gel, wells to be filled some with cells and others with chemoattractive molecules. During the experiment, the cells are free to migrate in response to the stimulus of the chemical gradient inside or else underneath the semisolid layer. In some variants, there are parallel wells and channels connected by a cut made right at the start of the experiment. In this way, it is possible to have available three or more channels and hence compare the chemotactic activity of different cell populations or else study which ligand^' is preferable within a group of interest. At the end of the experiment, it is possible to carry out in a non-automatic way the count of the cells that present a certain type of response to the stimulus. - Boyden chamber - multiwell (two-chamber technique) - the system known as Boyden chamber uses a hollow chamber made of plastic sealed at one end by a porous membrane. This chamber is suspended above a well of larger dimensions that may contain a culture medium and/or chemoattractive substances. The cells are positioned within the chamber and left to migrate within the pores to reach the other side of the membrane . At this point the migrating cells are marked and counted. In a standard Boyden assay, the diameter of the pores of the membrane is typically comprised between 3 and 12 μm, and is in any case selected on the basis of the size of the cells being tested. To a smaller size of the pores there corresponds a higher resistance to be overcome by the migrating cells. The majority of the cells has a size comprised between 30 and 50 μm and can migrate effectively through pores of 3-12 μm, whilst lymphocytes (having on average a dimension of 10 μm) are able to migrate also through pores in the region of 0.3 μm. 8-μm pores are suitable for migration of the majority of the epithelial cells and fibroblasts, but are unsuitable for experiments on the migration of lymphocytes .

The Boyden chamber exists also in a variant with a number of wells, which enables conduct of a number of experiments simultaneously. The cells that respond positively to the stimulus collect in the smaller chamber (for long incubation times) or else in the filter (for short incubation times) . For detection and counting of the cells standard staining techniques are used (for example, trypan blue) , or else techniques of marking with fluorochromes, or again recourse is had to specific probes . - Dunn chamber (two-chamber technique) - the chambers are connected horizontally (Zigmond chamber) or else are provided as concentric rings in a plane (Dunn chamber) , and are reclosed by a glass lid. A concentration gradient is developed bestride a narrow bridge that separates the chambers, and the cells are counted on the surface of the bridge by observation under the optical microscope. In some cases, the bridge between two chambers is filled with agar, and the cells that migrate in response to the concentration gradient are forced to traverse this semisolid layer.

Hence, the techniques proposed up to now, for example, also the ones according to the patent application No. US2003022153 that corresponds to a variant of the Zigmond chamber, impose laborious procedures and long times, entail very frequently the use of systems of specific markers (for example, based upon fluorescence) , require a very high number of manual operations and, consequently, cannot be reduced to an automatic procedure. In addition, they present obstacles to the possibility of operating with cells that are in a state that is as close as possible to the one that they would have in vivo and generally do not enable a fine adjustment of the experimental conditions. In particular, the average size of the pores through which the cells migrate in the chambers described previously is fixed beforehand; i.e., it cannot be modified in the course of a single experiment, but at the most between one experiment and the next. The Boyden chamber and the agar dish are moreover conceived for a single use and enable testing of a limited number of chemoattractive substances in a single experiment, the limit condition corresponding to a chamber in which it is possible to test a single chemoattractive substance at a time.

There hence exists the need in the art for systems for measuring chemotaxis that are more efficient and simpler than the ones currently in use and that preferably • enable automation.

Summary of the invention

Consequently, provided according to the present invention is a microfluidic system for making measurements of migration of cells, in particular, and of particles, in general, in response to application of a chemical gradient, said system enabling the aforesaid drawbacks to be overcome. In particular, according to the present invention a device is provided, comprising a plurality of reception sites for cells/particles and chemical compounds, according to what is defined in Claim 1.

In particular, according to the invention, the site that is to receive, for example via injection, the cells or particles and the at least one site, or plurality of sites, that is to receive, for example once again via injection, the chemoattractive/chemorepellent substances are fluidically connected to one another by a duct, the free section of passage of which results from the sum of the sections of passage of a plurality of micro-channels delimited by a corresponding plurality of obstacle elements set within the duct with a non-uniform density so as to create along the duct a gradient in the free section of passage at the disposal of the cells .

The obstacle elements can be elastically deformable . According to the invention, the free section of passage that results therefrom can, consequently, be modulated, in use, by operating appropriate deformation actuators from outside, hence enabling fine adjustment of the resistance that the cells must overcome to migrate by chemotaxis .

The same principle can be applied for modulating the chemical gradient, namely, by deforming a part of channel so as to enable optimal experimental conditions for the various types of chemoattractive substances being examined, which, in general, will have different coefficients of diffusion, according to their molecular weight and their interaction with the solution, which normally constitutes the culture medium of the cells.

Brief description of the figures

Further advantages and characteristics of the invention will emerge from the ensuing description of an embodiment thereof provided by way of non- limiting example, with reference to the attached drawings, wherein: Figure IA shows a schematic top plan view of a device according to the invention;

Figure IB shows a schematic top plan view of a multiwell variant of the device according to the invention; Figure 2A and 2B show perspective views of the obstacle elements that delimit and define the sections of passage of the ducts of the devices of Figures IA and IB;

Figure 3 shows a top plan view of a detail of a duct of the device of Figure IA or Figure IB;

Figure 4 shows a top plan view of a detail of the duct of Figure 3.

Detailed description

In Figure IA designated as a whole with 1 is a device according to the invention. Fluidically connected to a central site 2 are, by means of respective ducts 3 and 3', peripheral sites 4 and 4' . Likewise, Figure IB shows a variant of the device . 1 according to the invention comprising a single central site 2 fluidically connected to a plurality of peripheral sites 4 by a corresponding plurality of ducts 3.

According to one embodiment of the invention, the central site 2 is conceived for receiving a specimen of particles, for example a biological specimen basically constituted by cells. Here and in what follows, the terms "particles" or "particle" are meant to indicate micrometric or nanometric entities, whether natural or artificial, such as cells, subcellular components, viruses, liposomes, niosomes, microspheres and nanospheres, or even smaller entities, such as macro- molecules, proteins, DNA, RNA, etc.

Sometimes the term "cell" will be used, but, where not otherwise specified, it shall be understood as a non-limiting example of particles in the wider sense referred to above .

Here and in what follows, by the term "cell" or "cells" is understood both unicellular organisms and single cells of pluricellular organisms. More in general, the central cite 2 can receive generic particles, where by the term "particle" is understood, here and in what follows, a biological entity, such as cells, micro-organisms, viruses or parts thereof, for instance, portions of nucleic acids or proteins, plasmids, etc., or even a non-biological entity, such as a predetermined amount of a composite or substance, provided that it is in some way sensitive to chemotactic stimuli.

Each of the peripheral sites 4 is conceived for receiving a substance to be tested which is potentially capable of influencing chemotaxis . Said substance can comprise biomolecules, possibly immoblized. By "biomolecules" is understood, by way of non- limiting example, DNA, RNA, proteins, peptides, hydrocarbons, cells, chemical and biochemical compounds. In particular, the biomolecules are biomolecules capable of exerting on the cells of the specimen an attractive force greater than that of the forces naturally present in the fluidic medium around the specimen, such as those linked to solvatation and to turbulence.

By way of non- limiting example , the substance to be tested can be a chemorepellent substance , a chemotactic inhibitor, a chemoattractive substance , such as growth factors , cytokines , chemokines , nutrients , small molecules , and peptides .

Alternatively, the functions of the peripheral sites 4 and central sites 2 can be exchanged, in the case where it is intended to compare the behaviour of different cell populations - introduced each in distinct peripheral sites - in response to the chemical stimulus represented by the introduction in the central site of a single chemoattractive/repellent substance .

The sites 2 and 4 can be constituted, by wells made using any biocompatible material, from glass to a plastic, as likewise a substrate made of a semiconductor material, such as silicon, in which the wells can be made with different techniques common in the field of microcircuits, together, for example, as will be seen, with appropriate sensors and their accessory elements .

The ducts 3 , which can be made of the same material and in the same way as the sites 2, 4, according to a preferred embodiment of the device 1, are coplanar with the sites 2 and 4 and consist in channels with substantially rectangular right section as illustrated in Figures 2A and 2B.

Preferably, the sites 2 and 4 and the ducts 3 are made of a transparent material or provided with a transparent lid so that their contents can be observed easily under an optical microscope.

Contained within each duct 3, according to the invention, is a plurality of obstacle elements 5 in any case arranged with a non-uniform density so as to create along the duct 3 a gradient, either continuous or in steps (discrete) , in the free section of passage at the disposal of the cells.

In particular, the elements 5 are arranged, at a distance from one another by a regular or irregular pitch p, along an axis A that traverses substantially the centre of the central site 2 and the centre of the peripheral site 4 at the terminal end of the duct 3 itself. Each element 5 has a substantially "comblike" structure, defined by a plurality of substantially parallelepipedal "teeth" 5b set at a distance from one another, in a direction transverse to the axis A, by a distance d and hence defining between them a plurality of micro-channels, the openings of which have, precisely, a dimension d (see Figure 3) .

According to one embodiment of the invention, said dimension d decreases along the axis A, in the direction opposite to that of the concentration gradient (which comes to be created naturally, in use) of the chemoattractive substance so that the openings d defining the micro-channels will not generally be aligned in the direction of the axis A and will come to form, consequently, micro-channels which are not only with variable section of passage (progressively smaller in the direction opposite to that of the gradient of concentration) , but are also tortuous.

In practice, a cell 6 initially contained in the site 2 and exposed to a gradient of concentration of a chemoattractive substance (maximum concentration at the corresponding peripheral injection site 4 and decreasing along the axis A in the direction of the central injection site of the cell itself) will be induced to migrate towards the periphery of the system (in a direction indicated in Figure 3 by a vertical arrow directed upwards) and will encounter along the corresponding channel 3 the obstacle elements 5, 5' and 5" in sequence, the element 5' defining micro-channels of a size smaller than that of the channels defined by the element 5, and so on.

Since a greater intensity of the chemotactic effect on the cell results in a greater propension of the cell itself to deform in order to migrate through a micro-channel, the number of migrating cells that pass beyond elements 5 delimiting passages of progressively smaller dimensions is a parameter that provides information on the affinity of the cell for the chemoattractive substance being examined. Likewise, if comparative tests are carried out so that the cells 6 initially contained in the central site 2 are exposed simultaneously to concentration gradients of a number of chemoattractive substances Ci, C₂, ... C_n injected in respective peripheral sites 4, the greater or smaller degree of advance along a duct 3 corresponding to the chemoattractive substance Ci with respect to a distinct chemoattractive substance C_j will provide information on the relative affinity of the cell in regard to the different chemoattractive substances being examined .

The dimension d of the micro-channels is chosen as a function of the dimension of the cells being examined. Said dimensions d can be fixed for a given device, for example in so far as the "teeth" 5b are obtained, as shown in Figure 2A, on one and the same substrate (and with the same techniques) as the sites 2 and 4 and the ducts 3, or else, according to a preferred and further characterizing aspect of the invention, can be varied each time in one and the same device 1.

In this latter case, the obstacle elements 5 are not rigid elements, but are elastically deformable elements, for example made of a silicone resin, and it is possible, before carrying out the experiment, to select appropriate deformable elements 5 to cause them to assume a desired dimension d of the micro- channels, as will be seen.

According to a further embodiment (Figure 2B) , the obstacle elements 5, instead of being made integrally of a single piece with the same substrate in which the individual duct 3 is made can be made integral with a channel-shaped element 3b, which can be inserted in a removable way within the duct 3 , for example from above. In this case, the substrate in which the duct 3 is made can be defined by a plate 100, which bears also the sites 2 and 4, whilst the removable channel element 3b is made separately, even of a material different from that of the plate 100, and is inserted each time within each duct 3 before each experiment and removed at the end thereof. In this way, amongst other things, it is thus possible to provide a device 1 that can be reused but in which the material that comes into contact with the cells during the experiment can be removed and replaced, according to a "disposable" technique.

The dimension d can moreover be chosen on the basis of the particular chemoattractive substance used.

The size of the passages d can be, for example, 50, 10, 8, 7, 3, 0.5, 0.3 μm, according to the cells and the purposes of the experiments .

Preferably, inserted along a channel 3 are at least four deformable elements 5 having a dimension d of the micro- channels progressively decreasing so as to discriminate at least five thresholds of chemotactic migration.

The fabrication techniques comprise thin- film deposition and aquafortis etching, deep reactive-ion etching (DRIE) to obtain silicon masters, moulding of polymers, etc.

Starting from a silicon wafer coated with silicon dioxide, deposited by spin-coating is a photoresist layer, which is then thermally treated. There is then generated a pattern, and the oxide is etched. The^' silicon wafer is then etched by wet or dry etching to obtain the master mould for plastic moulding. The silicon master can finally be copied by means of nickel electrodeposition to obtain a stronger mould.

Once the bio-compatible flexible polymer has been moulded, the microfluidic device is completed by bonding or gluing thereto a flat bio-compatible flexible layer, or else by compressing the structure between two slides .

The basic concepts and the details of implementation of the photolithographic and etching techniques can ^' be found, for example, in "Thin film processes", J. Vossen, W. Kern, Academic Press, 1978, in T. J. Cotler, M. E. Elta: Plasma-etch technology, IEEE Circuits & Devices Mag. 6 (1990) 38-43 or else in J. L. Vossen, W. Kern, Thin film processes II, Academic Press, 1991.

According to the embodiment of the invention in which the obstacle elements 5 are deformable, around each of the channels 3 , in a region corresponding to one or more deformable elements 5, deformation actuators 7 can be provided, which, once operated, impose a more or less accentuated deformation of the elements 5, consequently- reducing the opening d of the corresponding micro-channels .

The same technique of "restriction" of the section of passage can be used also for another purpose.

It is known that the diffusion is generally regulated by equations of the Fick's-law type, as follows: dC d²C dt d x where C is the concentration of a substance and D its diffusion coefficient, which is a function of the size and shape of the molecules. Consequently, if it is possible to regulate appropriately the section of passage at the interface between the duct 3 and each site 4, for example, by acting on the side wall of the element 3b in the case where this and the corresponding "teeth" 5b are made of a deformable material, such as silicone, it would be possible to regulate appropriately the gradient of diffusion also when conducting the experiment.

This is obtained according to the invention by forming with the side wall of the element 3b in the proximity of a site 4 a sort of "valve" with variable opening and positioning and operating appropriate deformation actuators 7 in a region corresponding to said "valve" so as to modify the cross section of passage of the duct 3 that sets in connection the well 2 of the cells with the well 4 of the chemoattractive substance. In this way, the temporal course of the experiment can be finely adjusted taking into account the characteristics of the specific molecules (i.e., their coefficient of diffusion D) . For example, it is possible to operate so as to have the same space-time evolution of the concentration for different molecules by imposing

Ci(t,x) = C₂(t,x), with M₁ ≠ M₂ (2)

where M₁ and M₂ are, respectively, the molecular weights of the molecules 1 and 2, x is the spatial co-ordinate, which in general is a vector in a three-dimensional space, and the concentration Ci is expressed as number of molecules per unit volume .

Hence, in the case of an element 3b made of a deformable material, both the width of the micro-channels d and the width of the overall section of passage of the duct 3 at the interface with the site 4 can in effect be finely adjusted by compressing or elongating the elements 5 and/or the side wall of the element 3b, which can be deformed by means of the external actuators 7. Figure 4 illustrates by way of example an actuator 7 operated to bring about deformation of the wall of the duct 3 and consequent reduction of the opening of the micro-channel adjacent to the wall to a transverse dimension d" smaller than the pre-set starting transverse dimension d. Preferably, for said purpose piezoelectric bimorph actuators are used, of a type substantially known and consequently not described in detail for reasons of simplicity. Preferably, the device is moreover provided with appropriate control means 8 (Figure 3) for regulating the degree of deformation imposed by the actuators 7 on the walls of the ducts 3. In each channel two distinct areas controlled by respective actuator means may be identified, one for the formation of the cell gradient and one for controlling the diffusion of the chemoattractive substance and of the corresponding chemical gradient. The dimensions of the "sieves" may be different, from 0.3 to 15 μm for the micro-channels for formation of the cell-concentration gradient and from one nanometre to some micrometres for the chemical gradient, according to the substance used.

It is moreover possible to implement automatic-counting systems, such as:

- transparent electrodes (for example, ITO electrodes) : these can be used for automatic counting of the cells that migrate from different sites by means of measurements of electrical impedance ;

- micro-balances: these can be used for automatic counting of the cells that migrate from different sites by means of measurements of mechanical impedance; for example, a thin-film piezoelectric actuator can be integrated in a duct 3 : its resonance frequency will be affected by the presence of cells on its surface.

In general, therefore, the device 1 of the invention can comprise means 9 designed to detect the presence of cells within the duct 3, which are possibly also constituted by detectors of an optical type. In addition, the device 1 can be provided with concentration sensors 10 capable of detecting the point concentration or else the evolution of the concentration gradient, of the chemoattractive substance along the duct 3.

According to an advantageous aspect of the invention, said detection means 9 and sensors 10 are appropriately connected with an external processing unit 11 to which they are designed to transmit, by wired or radiofrequency transmission means, the values of the quantities detected for their processing, display, recording, etc.

Experimental methodology The cells introduced are suspended in a culture medium. The concentration is regulated according the type of cells. Since the exposed area of the well is comprised between 5 and 100 mm², a suspension of 10 000-50 000 cells in 1000-2000 microlitres is used. For example, in a single dish (defining a site 2) 20 000 macrophages should be introduced.

After the central well (site 2) has been filled with the cell suspension, the peripheral wells (sites 4) are filled with the cell-culture medium containing the solution of the chemotactic factor (or control solution, if the chamber functions as negative control) . For example, for the analysis of chemotaxis of the macrophages, 20 nM C5a of chemotactic peptide should be used.

Preferably, the solutions are preliminarily heated (37⁰C) and degassed to prevent bubbles from appearing in the course of incubation. Care should moreover be taken not to touch the chamber (the device 1) with bare hands.

Next, the chamber is incubated for the time necessary, and the migrating cells are electronically counted. For the majority of the chemotaxis assays this operation is executed at 37⁰C in humidified air with 5% of CO₂. The optimal incubation time can vary considerably according to the type of cell and of chemotactic factor. For macrophages, the continuous measurement of migrating cells must be monitored for four hours .

Unlike what happens in a Boyden chamber or a Dunn chamber, the device of the invention enables measurement of cell migration in an automatic way. In addition, the use of the device of the invention enables an estimate to be obtained of the cell speed and of the directionality of the cell motion that do not depend upon the subjectivity of the operator.

In the multiwell embodiment of the device it is also possible to conduct assays in competition between different stimuli. In addition, the assay conditions can be modified in the course of the experiment, for example by altering the degree of opening of one of the ducts 3 or else by closing completely one of them so as to exclude the contribution of a chemoattractive molecule from the overall chemotactic effect on the cells, whereas in the apparatuses known as Boyden and Dunn chambers it is impossible to modulate the experimental conditions once the assay has started.

Claims

1. A device (1) for measuring the migration of particles, for example cells, in response to exposure to a gradient of chemical concentration, comprising a first site (2) , designed to receive a specimen of particles, and at least one second site (4) , designed to receive a substance potentially capable of influencing chemotaxis, said first and second sites (2, 4) being fluidically connected together by a duct (3) , said device being characterized in that said duct contains, set along an axis A directed between said first and second sites (2, 4), a plurality of obstacle elements (5) with a nonuniform density so as to create along the duct a gradient in the free section of passage at the disposal of the cells defined by a corresponding plurality of micro-channels directed parallel to the axis A and having a pre-set transverse dimension (d) .

2. The device (1) according to Claim 1, characterized in that said obstacle elements (5) have a substantially comblike structure and are formed by a plurality of teeth (5b) set alongside one another defining between them respective openings (5c) forming the micro- channels .

3. The device (1) according to Claim 1 or Claim 2, characterized in that said pre-set transverse dimension (d) decreases along said axis A in the increasing sense of said concentration gradient .

4. The device (1) according to any one of Claims 1 to 3 , characterized in that said obstacle elements (5) are made integrally of a single piece with the same substrate in which said duct (3) is made.

5. The device (1) according to any one of Claims 1 to 3 , characterized in that said obstacle elements (5) are made integrally of a single piece with a channel-shaped element (3b) , which can be inserted in a removable way within the duct (3) .

6. The device (1) according to any one of Claims 1 to 5, characterized in that it comprises a plurality of said second sites (4) , each fluidically connected to said first site (2) by a respective duct (3) .

7. The device (1) according to any one of Claims 1 to 6 , characterized in that it is made of a transparent material .

8. The device (1) according to any one of the preceding claims, characterized in that said obstacle elements (5) are made of a deformable material .

9. The device (1) according to Claim 8, characterized in that said obstacle elements (5) are made of an elastically deformable material.

10. The device (1) according to Claim 8 or Claim 9, characterized in that it moreover comprises a plurality of actuator means (7) for reducing the dimension d of said micro- channels .

11. The device (1) according to Claim 10, characterized in that said ducts (3) are delimited by walls made of an elastically deformable material, said actuator means (7) being each designed to deform the walls of a corresponding duct (3) so as to deform at the same time one or more of the deformable elements (5) contained in said duct (3) .

12. The device (1) according to Claim 11, characterized in that it comprises control means (8) , designed to regulate the degree of deformation imposed on the walls of the duct (3) by said actuator means (7) .

13. The device (1) according to any one of Claims 10 to 12, characterized in that it comprises first and second actuator means (7) arranged so as to form, along each of said ducts (3) , two distinct areas controlled, a first one by the first actuator means and a second one by the second actuator means, the first area to obtain formation of a cell gradient and the second area to obtain control of the diffusion of the chemoattractive substance and of the corresponding chemical gradient .

14. The device according to any one of Claims 10 to 13 , characterized in that said actuator means (7) are bimorph piezoelectric actuators .

15. The device according to any one of the preceding claims, characterized in that it comprises means (9) designed to detect the presence of cells within said duct (3) .

16. The device according to Claim 15, characterized in that said detection means (9) comprise electrodes for impedance- metric measurements, piezoelectric microbalances, optical detectors .

17. The device according to any one of the preceding claims, characterized in that it comprises sensor means (10) , designed to detect the point concentration and the gradient of chemoattractive substance along said duct (3) .

18. The device according to any one of Claims 15 to 16, characterized in that said detection means (9) are provided with wired or radiofrequency transmission means for transmitting the quantities detected to a processing unit.

19. The device according to Claim 17, characterized in that said sensor means (10) are provided with wired or radiofrequency transmission means for transmitting the quantities detected to a processing unit (11) .