Device for monitoring the growth of a three-dimensional cell cluster and method for manufacturing the same

Abstract

Device (1), and method for manufacturing the same, for detecting the growth of a three-dimensional cell cluster (C), comprising: a substrate (2) having a support surface (21); a plurality of electrodes (3) arranged according to a predetermined spatial distribution, for detecting an electrical potential in the presence of a three-dimensional cell cluster (C); a plurality of three-dimensional scaffolds (4), comprising at least a first scaffold (41), arranged at a respective measuring electrode (31), and at least a second scaffold (42), arranged at a respective reference electrode (32), wherein the at least one first scaffold (41) has a proliferation conformation, adapted to promote the growth of the three-dimensional cell cluster (C), and the at least one second scaffold (42) has an inhibition conformation adapted to prevent the growth of the three- dimensional cell cluster (C).

Description

[0001] TITLE: “Device for monitoring the growth of a three-dimensional cell cluster and method for manufacturing the same”

[0002] DESCRIPTION

[0003] FIELD OF APPLICATION

[0004] The object of the present invention finds useful use in the biotechnology sector, in molecular biology and in regenerative medicine. In particular, it finds useful application in the study of cellular behaviour in relation to drugs and cytotoxic factors.

[0005] DESCRIPTION OF THE PRIOR ART

[0006] It is known to the state of the art to realize devices for monitoring the growth of a cell cluster and related systems for performing cell impedance measurements.

[0007] In detail, the known devices that monitor cellular behaviour comprise a substrate on which a two-dimensional array of measuring and reference electrodes is integrated. On said devices, scaffolds are realized which are adapted to accommodate a cell culture to allow and perpetuate its growth. The electrode array is electrically connected to an impedance analyser, to detect the impedance variations between the measuring electrodes and the reference electrodes due to the influence of drugs or cytotoxic factors on the cells of the cell cluster. In fact, the cells of the cluster adhere on the surface of the substrate and, by effect of the insulating properties of the cell membrane, create a resistance to the passage of current between the electrodes, resulting in an increase in potential between the measuring electrode and the reference electrode. Therefore, the measurement of impedance, calculated in relation to cell adhesion, proliferation, growth and morphology, is an accurate methodology for performing estimates of the behaviour of the cell cluster examined.

[0008] Problems of the prior art

[0009] The devices known to the state of the art have several limitations. In particular, most of the known devices are limited to the cultivation and consequent measurement of two-dimensional cell cultures, which do not allow the biological complexity of a three-dimensional cell mass to be appropriately modelled, thus obtaining estimates that are not entirely faithful to real cases.

[0010] Furthermore, the probability of obtaining cell clusters adhering to the two- dimensional electrode array is significantly lower and, consequently, this significantly impacts the impedance growth signal, consequently leading to an imprecise evaluation of the cell behaviour.

[0011] Additionally, in the two-dimensional cell clusters, the cells are free to migrate within the scaffold without appropriate guidance, thereby causing cell proliferation in areas of the device which, in addition to not being of interest, negatively affect the impedance measurement and overall evaluation.

[0012] SUMMARY OF THE INVENTION

[0013] The object of the invention is to make available a device for monitoring the growth of a three-dimensional cell cluster capable of overcoming the limitations of the prior art. In particular, it is an object of the present invention to provide a device for monitoring the growth of a three-dimensional cell cluster in such a way as to allow an accurate measurement and a faithful estimate of the cell behaviour.

[0014] A further object of the present invention is to provide a method for realizing the aforementioned device.

[0015] Still, it is an object of the present invention to provide a system and related method for measuring the impedance variations, in relation to cellular behaviour, detected by the aforementioned device.

[0016] These and further purposes will be substantially achieved by a device, a method and a system having the technical characteristics described in one or more of the appended claims.

[0017] Advantages of the invention

[0018] Thanks to the present invention it is possible to make available a device capable of monitoring the growth and the development of three-dimensional cell clusters, organoids and / or spheroids, in order to monitor the formation of cell tissue and estimate the cell behaviour accurately and more precisely than the prior art. In fact, the device of the present invention promotes cell growth at the measuring electrodes and inhibits it at the reference electrodes.

[0019] Advantageously, in this way, the impedance analysis of the examined three- dimensional cell cluster is facilitated and refined. Consequently, such analysis can serve as an advanced high-throughput screening approach for the study of new drugs and therapies and for the study of the natural evolution of the three-dimensional cell cluster. BRIEF DESCRIPTION OF THE DRA WINGS

[0020] The advantages and the technical characteristics will be made abundantly clear by the following detailed description of a device for monitoring the growth of three- dimensional cell clusters and a method for realizing the same, in accordance with a preferred and therefore non-limiting embodiment, in the set of drawings wherein:

[0021] - figure la shows a device for monitoring the growth of a three-dimensional cell cluster according to the present invention in accordance with an embodiment;

[0022] - figure lb shows a temporal succession of steps of realization of the device of figure 1;

[0023] - figure 2a shows a schematic temporal sequence of a step of the method of the present invention;

[0024] - figure 2a shows a top view of a detail of the step of the method shown in figure 2a;

[0025] - figure 2b shows a top view of a further detail of the step of the method shown in figure 2a;

[0026] - figure 3 shows a perspective view of a detail of the device of figure 1;

[0027] - figure 4 shows a perspective view of a detail of the device of the present invention;

[0028] - figure 5a shows a front view of the detail of figure 4;

[0029] - figure 5b shows a side view of the detail of figure 4; - figure 5c shows a top view of a detail of the item of figure 4;

[0030] - figure 5d shows a bottom view of the detail of figure 4,

[0031] - figure 6 shows a block diagram of the system according to the present invention.

[0032] DETAILED DESCRIPTION

[0033] As shown in figure 1, reference numeral 1 indicates a device for detecting the growth of a three-dimensional cell cluster C.

[0034] In the context of the present invention, “three-dimensional cell cluster” means a three-dimensional multicellular structure such as organoid and / or spheroid, whose dimensions can range from tens of micrometres to tens of millimetres.

[0035] The device 1 of the present invention comprises a substrate 2 of thermally stable and chemically inert material, preferably glass, having a support surface 21. As will be seen below, these properties advantageously allow the chemical-physical structure of the substrate 2 to be maintained unchanged during the manufacturing steps of the device 1 described in the following of this description.

[0036] Preferably, the substrate 2 has dimensions in the order of tens of millimetres and a thickness in the order of millimetre units. In the preferred form, the substrate 2 has a dimension of 76 x 26mm and a thickness of 1mm.

[0037] The device 1 comprises a plurality of electrodes 3. The plurality of electrodes 3 in turn comprises at least one measuring electrode 31 and at least one reference electrode 32. In the device 1 of the present invention the plurality of electrodes 3 is integrated on the support surface 21 of the substrate 2. Advantageously, this allows to avoid using reference electrodes that are not integrated in the device 1 itself, i.e. external ones, facilitating the use of the device 1 and improving the reproducibility of the measurement data obtained.

[0038] The plurality of electrodes 3 is further arranged according to a predetermined spatial distribution. This predetermined spatial distribution can be realized according to different arrangements, in relation to the needs required by the specific application selected by the technician in the sector.

[0039] It should be noted that, in the embodiments in which the plurality of electrodes 3 comprises at least two measuring electrodes 31 and at least two reference electrodes 32, the assumed spatial distribution may have a regular pattern repeated in space.

[0040] Note that the term “pattern” refers to a model or a specific configuration used to designate a diagram of an integrated circuit.

[0041] In accordance with the invention, the plurality of electrodes 3 is configured to detect the electric potential, when a three-dimensional cell cluster C is cultivated on the support surface 21.

[0042] In other words, the plurality of electrodes 3 is configured to detect the electric potential between the at least one measuring electrode 31 and the at least one reference electrode 32 in the presence of a flow of electric current. In particular, the presence of the three-dimensional cell cluster C, by effect of the insulating properties of the cell membrane, hinders the passage of this flow of current and causes the variation of potential. Hence, the three-dimensional cell cluster C simulates the behaviour of an electrical resistance to the passage of the flow of current between the at least one measuring electrode 31 and the at least one reference electrode 32. Therefore, the presence of cytotoxic factors or drugs has an effect on cell growth, migration and adhesion, consequently causing a variation in the impedance generated by the three- dimensional cell cluster C and in the electrical potential measured.

[0043] It should be noted that the plurality of electrodes 3 also comprises the electrical connections necessary for their operation in detecting the electrical potential (not shown in detail in the accompanying figures).

[0044] The device 1 further comprises a plurality of three-dimensional scaffolds 4, arranged in accordance with the predetermined spatial distribution of the plurality of electrodes 3.

[0045] In particular, the plurality of three-dimensional scaffolds 4 comprises at least a first scaffold 41, arranged at a respective measuring electrode 31, and at least a second scaffold 42, arranged at a respective reference electrode 32.

[0046] Note that the number of the first scaffolds 41 corresponds to the number of measuring electrodes 31, and similarly, the number of second scaffolds 42 corresponds to the number of reference electrodes 32.

[0047] In the device 1, the at least one first scaffold 41 has a proliferation conformation, adapted to promote the growth of the three-dimensional cell cluster C, and the at least one second scaffold 42 has an inhibition conformation adapted to prevent the growth of the three-dimensional cell cluster C.

[0048] In other words, the at least one first scaffold 41 has a conformation such as to allow the spreading and growth of the three-dimensional cell cluster C, unlike the at least one second scaffold 42, which has a conformation, substantially different from that of the at least one first scaffold 41, adapted to hinder the growth and proliferation of the three-dimensional cluster C.

[0049] Advantageously, this allows to direct and promote cell growth at the measuring electrodes 31 and at the same time to leave the reference electrodes 32 free, thus allowing to perform an accurate and precise measurement of the electrical pulses emitted by the cells.

[0050] According to the invention, the proliferation conformation is a dome structure, preferably designed by using Blender software. In other words, the proliferation conformation has a shape substantially like a spherical cap. Preferably, the dome structure has a diameter of 300pm.

[0051] In alternative embodiments, the proliferation conformation may have structures having, externally, the geometry of a regular polyhedron, such as for example dodecahedron and icosahedron, of an archimedean polyhedron, such as for example cuboctahedron and icosidodecahedron, but also of a prism and antiprism, or of a stellated polyhedron, such as for example the large icosahedron. Preferably, regardless of the geometry used, the proliferation conformation has a polyhedron structure sectioned at the base, i.e. sectioned at the at least one measuring electrode 31. Advantageously, in this way, a more rapid colonization of the structure by the cells of the three-dimensional cell cluster C is allowed.

[0052] According to the invention, the dome structure has a cavity (not shown in the accompanying figures) adapted to accommodate the three-dimensional cell cluster C to allow its proliferation. As shown in the embodiment of figure 3, the dome structure comprises a plurality of polygonal windows 412, preferably pentagonal and / or hexagonal windows.

[0053] These polygonal windows 412 preferably have a conformation and an extension such as to be able to allow both cell seeding within the cavity of the dome structure, and the entire colonization of the first scaffold 41, including the entire dome structure.

[0054] In more detail, in the embodiment shown in figure 3, the dome structure comprises a plurality of hexagonal windows Al -A3, a first and a distinct second plurality of pentagonal windows A4-A6, A7-A9. Note that, in the present embodiment, the windows Al, A4 and A7 are the outermost windows, the windows A2, A5, A8 are the intermediate windows, and the windows A3, A6 and A9 are the innermost windows.

[0055] An example of the dimension of the respective windows is reported in Tables 1, 2 and 3:

[0056] Table 2

[0057] Table 3

[0058] According to the invention, unlike the proliferation conformation of the first scaffold 41, the inhibition conformation of the second scaffold 42 has a tower structure.

[0059] Advantageously, in this way, the tower structure allows to guarantee a protection function in such a way that the growth of the three-dimensional cell cluster C is limited at the second scaffolds 42.

[0060] In an embodiment shown in the accompanying figures 4, 5a, 5d, the tower structure of the inhibition conformation comprises a baseplate 421, preferably conformed to a parallelogram with a square base, preferably with a side equal to 300pm. As shown in accompanying figure 5d, the baseplate 421 has a plurality of walls, preferably with thickness equal to 36pm. The tower structure of the inhibition conformation preferably has a prevalent extension oriented along a vertical direction Z-Z equal to 720pm. With reference to figure 5a, this extension is defined by the aforementioned base 421 and by a plurality of columns 422, wherein each column is distinct from the adjacent column and separated therefrom by a predetermined distance.

[0061] In an embodiment shown in figure 5c, each column has a cylindrical geometry, preferably with a diameter equal to 20.5pm, and also has a prevalent extension along the vertical direction Z-Z, preferably equal to 240pm.

[0062] In the accompanying figures 4, 5a-5b, the tower structure further comprises a roof 423, preferably sloping with two pitches, defining a gable. Preferably, the gable has a base of 400pm and a height of 88pm.

[0063] In alternative embodiments, the inhibition conformation has a mushroom structure having a plurality of pores, or a hedgehog structure, with pointed spines that prevent cell growth over the at least one reference electrode 32. Still alternatively, the inhibition conformation may provide for a multi-spined porous membrane structure.

[0064] According to a further embodiment, the inhibition conformation comprises porous structures with non-adhesive material for the cells of the three-dimensional cell cluster C manufactured above and around the at least one reference electrode 32.

[0065] According to a preferred aspect, the proliferation conformation and / or the inhibition conformation, preferably both, have a plurality of porous walls. With greater reference to the inhibition conformation, the plurality of porous walls preferably have a specific structure in such a way as, in combination with the tower structure, to limit the growth of the three-dimensional cell cluster C and, at the same time, still allow the passage of current through the electrodes in such a way as to perform the measurements.

[0066] In other words, in the case of the tower structure, the entry of the cells of the three-dimensional cell cluster C is mainly inhibited by the sloping roof 423, which prevents the entry of the cells from above during cell seeding, and the baseplate 421, which prevents the colonization of the cells after seeding. Actually, it should be specified that the baseplate 421 has a plurality of non-porous walls having an extension along the vertical direction Z-Z of 352pm.

[0067] It should also be noted that, the plurality of columns 422 have a structural function, as they are configured to act as a support for the roof 423, allow the passage of electric current between the empty spaces, and, finally, significantly reduce the probability that some cells during seeding will be able to enter the tower structure in the event of small oscillations or vibrations, due for example to the movement of the device 1 in an incubator or under the microscope.

[0068] A further object of the present invention is a method for manufacturing the device 1.

[0069] In particular, the method comprises the steps of a) providing the substrate 2. Subsequently, the method comprises the step of b) realizing the plurality of electrodes 3 integrated on the support surface 21 of the substrate 2 and arranged according to a predetermined spatial distribution by means of photolithography techniques. Preferably, step b) also provides for realizing, together with the plurality of electrodes 3, also the electrical connections necessary for their operation, i.e. for detecting the electrical potential. Finally, the method provides for c) realizing, by means of photolithography techniques, the proliferation conformation of the at least one first scaffold 41 at the at least one measuring electrode 31, and the inhibition portion of the at least one second scaffold 42, at the at least one reference electrode 32.

[0070] By way of example, figure lb shows a time sequence relating to the formation of the proliferation structure of a first scaffold 41 on a measuring electrode 31.

[0071] According to a preferred form, the step of c) realizing, by means of photolithography techniques, the proliferation conformation of the at least one first scaffold 41 at the at least one measuring electrode 31, and the inhibition portion of the at least one second scaffold 42 at the at least one reference electrode 32 provides for deriving a positioning map by defining the coordinates of the plurality of electrodes 3 arranged according to the predetermined spatial distribution.

[0072] Subsequently to the step of generating a positioning map, the step of c) realizing provides for arranging the plurality of three-dimensional scaffolds 4 on the support surface 21 of the substrate 2 in accordance with the positioning map generated in the previous step, such that the at least one first scaffold 41 is positioned at the at least one measuring electrode 31 and the at least one second scaffold 42 is positioned at the at least one reference electrode 32.

[0073] In the preferred form, the step of c) realizing the proliferation conformation and the inhibition conformation provides for manufacturing the plurality of three- dimensional scaffolds 4 by means of multi-photon polymerization technique. Preferably, the plurality of three-dimensional scaffolds 4 is made of non-toxic IP-S resin, a material suitably suitable for processing by means of polymerization techniques. Advantageously, multi-photon polymerization makes it possible to realize three-dimensional structures inside a predetermined volume of resin and the possibility of realizing structures with a resolution of up to lOOnm.

[0074] Still preferably, the step of c) realizing provides for developing the proliferation and inhibition structures of the at least one first and second scaffolds 41, 42 by means of propylene glycol methyl ether acetate (PGMEA) for 5 minutes, followed by a washing step with isopropyl alcohol for 15 minutes.

[0075] An embodiment, schematically shown in figure 2a, relating to the step b) for realizing the plurality of electrodes 3 on the substrate 2 support surface 21 will now be described.

[0076] According to this preferred form, the step b) of realizing provides for:

[0077] I) coating the support surface 21 of the substrate 2 with a first resist by means of coating techniques, such as for example spin coating technique. Note that the term “resist” or “photoresist” means a polymeric material sensitive to light radiation.

[0078] Step I) preferably provides for cleaning the support surface 21 itself by means of deionized water, acetone and isopropanol and subsequently drying it by means of an air drying system. Subsequently, the support surface 21 of the substrate 2 can be treated by means of a plasma activation technique to improve its adhesion properties.

[0079] Preferably, the first resist is of the negative type, such as for example AZ LNR 003. In other words, a predetermined amount, preferably 2ml, of the first resist is deposited at the centre of the support surface 21 of the substrate 2. The latter is subsequently placed on a rotating disk which, by effect of the rotation, preferably at a rotation speed equal to 2300rpm for 35s, allows the resist to be laid on the entire support surface 21 of the substrate 2. Subsequently, the same step provides for subjecting the substrate 2 to a controlled heating process on a plate at 120 °C for 2 minutes.

[0080] II) realizing, by means of the deposition of a first photographic mask Ml, a spatial distribution on the support surface 21, in the presence of a light source, such as, for example, a UV ray source at a dose of 350mJ / cm2.

[0081] It should be noted that a photographic mask has transparent regions and complementary opaque regions, respectively adapted to allow and hinder the passage of light. Being known to a person skilled in the art, the photographic masks will not be described further in the following of this description. In particular, preferably, the arrangement of the first photographic mask Ml provides for aligning the latter by means of a mask aligner. After exposure to the light source, the deposition of the first photographic mask Ml provides for placing the support surface 21 of the substrate 2 at a temperature of 100 °C for 90 seconds.

[0082] Step II) relating to the deposition of the first photographic mask Ml provides for depositing a developer, such as for example AZ 726 MIF for 60 seconds, in such a way that the latter acts on the first resist to complete the realization of the spatial distribution. The developer can then be removed by rinsing with deionized water.

[0083] In other words, the deposition of the photographic mask allows the external light source to irradiate only those regions of the support surface 21, having the first resist, corresponding to the transparent regions of the photographic mask itself. In this way, the light emitted, by irradiating the first resist of the negative type, causes the reinforcement of the material following a polymerization or cross-link process. Consequently, the developer, by acting on the first resist itself, dissolves the regions not hit by the light emitted by the resist, thus creating the spatial distribution on the first resist deposited on the support surface 21 of the substrate 2.

[0084] III) making the spatial distribution, obtained in the previous step, conductive by depositing at least one layer of metallic material on the support surface 21 coated by the resist. Preferably, the at least one layer provides for depositing a first layer of titanium and a second layer of platinum using Argon. Preferably, the first layer of titanium and the second layer of platinum have a thickness of lOnm and 150nm, respectively.

[0085] IV) removing the first resist from the support surface 21 of the substrate 2, preferably by means of acetone and isopropanol.

[0086] V) depositing a protective layer, preferably made of Parylene C, on the support surface 21 of the substrate 2, having the metallic spatial distribution, by means of the thermal deposition techniques. According to one aspect of the invention, the protective layer has a thickness of 500nm.

[0087] In alternative embodiments, it is possible to deposit protective layers made of Benzocyclobutene (BCB) or Polyimide, which however have higher costs and processing temperatures (above 200 °C) that can compromise the device 1. Differently, in terms of optical, electrical properties, costs, biocompatibility and techniques of use, Parylene C is the most advantageous in that deposition takes place by means of thermodeposition techniques at a temperature below 100 degrees without damaging the device 1. VI) coating the support surface 21 having the protective layer of the previous step with a second resist by means of coating techniques. Preferably, the second resist is of the positive type, such as for example AZ 10 TX. Similarly to the first resist, the second resist is extended in its entirety on the support surface 21, having the conductive spatial distribution, by means of preferably spin coating techniques. In other words, an amount, preferably equal to 2ml, of the second resist is deposited in the centre of the support surface 21, having the conductive spatial distribution. Subsequently, the entire substrate 2 is positioned on the rotating disk, having a rotation speed equal to 3000rpm for 35s, in motion such as to spread the second resist over the entire extension of the support surface 21 of the substrate 2, and then heated to a temperature of 110 °C for 2 minutes.

[0088] VII) realizing, by means of the deposition of a second photographic mask M2, a spatial distribution on the support surface 21 coated by the second resist in the presence of the light source, preferably by UV source at a dose of 1500mJ / cm2.

[0089] Preferably, the first and second photographic masks Ml, M2 are realized by means of LibreCad software.

[0090] Similar to the first photographic mask Ml, the second photographic mask M2 is aligned with respect to the support surface 21 by using a mask aligner.

[0091] In particular, as can be seen in figures 2b-2c, the second photographic mask M2 is opposite to the first photographic mask Ml. In other words, the transparent regions of the first photographic mask Ml correspond to the opaque regions of the second photographic mask M2. Consequently, the second photographic mask M2 has the transparent regions at the conductive spatial distribution arranged on the support surface 21. After light exposure, step VII) further provides for depositing the developer described above for a time equal to 5 minutes. Note that, the second positive resist acts differently with the developer, compared to the first negative type resist. Actually, the regions of the second resist of the positive type, irradiated by light, are degraded. Consequently, the developer only dissolves those regions of the second resist that have been irradiated by the light beam, i.e. the regions at the conductive spatial distribution. Therefore, following the step just described, the same spatial distribution is formed on the second resist.

[0092] Following application of the developer, the substrate 2 is rinsed with deionized water and dried with a flow of N2.

[0093] VIII) working the protective layer at the spatial distribution resulting from the previous step, by means of etching techniques, such as for example Oxygen plasma etching, preferably for a time equal to 5 minutes at 100W.

[0094] IX) remove the remaining second resist by acetone. This step provides for rinsing the substrate 2 with isopropanol.

[0095] At the end of the succession of the steps described above, the support surface 21 of the substrate 2 has a plurality of electrodes 3 (conductive spatial distribution) integrated.

[0096] According to one embodiment, the step of generating a map by defining the coordinates of the plurality of electrodes 3 arranged according to the predetermined spatial distribution provides for detecting the variations of the optical interface between the protective layer, preferably Parylene C, and the second resist. In fact, the optical interface variations cannot be detected at the electrodes as the optical interface variations between the latter and the resist would be extremely high. For this reason, the interface can be identified at a predetermined distance from each electrode and, by software processing, it is possible to program a positioning of the scaffold at the same distance in the opposite direction. It is therefore possible to construct a positioning map by exploiting the relative distance between the electrodes and automatically program the positioning of the plurality of three-dimensional scaffolds 4 on the plurality of electrodes 3 by finding the optical interface on the side of each electrode of the plurality of electrodes 3.

[0097] A further object of the present invention is a device 1 obtained by the method described above. The device obtained with the aforementioned method comprises at least a first scaffold 41 having a proliferation conformation, adapted to promote the growth of the three-dimensional cell cluster C, and at least a second scaffold 42, having an inhibition conformation adapted to prevent the growth of the three-dimensional cell cluster C.

[0098] As shown in figure 6, it is also an object of the present invention a system 10 for performing interferometric measurements of a three-dimensional cell cluster C. The system 10 comprises the device 1 described above, a stimulator 101 comprising a plurality of stimulation electrodes (not shown in the accompanying figures) configured to deliver a flow of electric current through the three-dimensional cell cluster C.

[0099] The system 10 further comprises an impedance analyser 102, in signal communication with the plurality of electrodes 3. The impedance analyser 102 is configured to measure the variation of electric potential between the at least one measuring electrode 31 and the at least one reference electrode 32, in the presence of the flow of electric current. It should be noted that the impedance analyser 102 is suitably connected to the plurality of electrodes 3 by means of the aforementioned electrical connections (not shown in detail in the accompanying figures).

[0100] In this way it is possible to evaluate the cellular behaviour in relation to a plurality of cytotoxic factors or drugs. In particular, following the influence of the latter, the impedance due to the presence of the three-dimensional cell cluster C varies, in turn varying the potential difference measured between the at least one measuring electrode 31 and the at least one reference electrode 32. By monitoring the aforesaid potential variations, it is therefore possible to evaluate the cellular impedance and consequently estimate the behaviour of the three-dimensional cell cluster C.

[0101] Finally, a method for performing interferometric measurements of a three- dimensional cell cluster C is an object of the present invention, comprising the following steps of providing the system described above. Subsequently, the method provides for cultivating a three-dimensional cell cluster C on the support surface 21.

[0102] Preferably, the step of cultivating a three-dimensional cell cluster C on the support surface 21 provides for promoting the growth of the three-dimensional cell cluster C by inserting the device 1 inside a culture chamber (not shown in the accompanying figures), preferably sized 1cm x 1cm.

[0103] Finally, the method comprises the step of measuring the impedance variations, by means of the impedance analyser 102, of the three-dimensional cell cluster C in a predetermined time window, such as for example 24, 48 or 96 hours, in the presence of the flow of electric current generated by the stimulator 101.

Claims

CLAIMS1. Device (1) for detecting the growth of a three-dimensional cell cluster (C), comprising:- a substrate (2) of thermally stable and chemically inert material, having a support surface (21);- a plurality of electrodes (3) comprising at least one measuring electrode (31) and at least one reference electrode (32), the plurality of electrodes (3) being integrated on the support surface (21) of the substrate (2) and arranged according to a predetermined spatial distribution, the plurality of electrodes (3) being configured to detect an electrical potential in the presence of a three-dimensional cell cluster (C) when cultivated on the support surface (21);- a plurality of three-dimensional scaffolds (4), arranged in accordance with the predetermined spatial distribution of the plurality of electrodes (3), and comprising at least a first scaffold (41), arranged at a respective measuring electrode (31), and at least a second scaffold (42), arranged at a respective reference electrode (32); characterized in that the at least one first scaffold (41) has a proliferation conformation adapted to promote the growth of the three-dimensional cell cluster (C), and the at least one second scaffold has an inhibition conformation adapted to prevent the growth of the three-dimensional cell cluster (C), wherein the proliferation conformation is a dome structure having a cavity adapted to accommodate the three- dimensional cell cluster (C) and the inhibition conformation has a tower structure.

2. Device (1) according to claim 1, wherein the dome structure comprises a plurality of polygonal windows (411), preferably pentagonal and / or hexagonal windows.

3. Device (1) according to any of the preceding claims, wherein the proliferation conformation and / or the inhibition conformation have porous walls.

4. Method for manufacturing the device (1) according to any one of claims 1 to 3, comprising the step of: a) providing the substrate (2); b) realizing the plurality of electrodes (3) integrated on the support surface (21) of the substrate (2) and arranged according to a predetermined spatial distribution by means of photolithography techniques; c) realizing, by means of photolithography techniques, the proliferation conformation of the at least one first scaffold at the at least one measuring electrode (31), and the inhibition portion of the at least one second scaffold (42) at the at least one reference electrode (32).

5. Method according to claim 4, wherein the step of c) realizing provides for:- deriving a positioning map by defining the coordinates of the plurality of electrodes (3) arranged according to the predetermined spatial distribution;- arranging the plurality of three-dimensional scaffolds (4) on the substrate (2) in accordance with the positioning map generated in the previous step, such that the at least one first scaffold is positioned at the at least one measuring electrode (31) and the at least one second scaffold (42) is positioned at the at least one reference electrode (32).

6. Method according to claim 4 or 5, wherein the step of c) realizing, by means of photolithography techniques, the proliferation conformation and the inhibition conformation provides for manufacturing the plurality of three-dimensional scaffolds (4) by means of multi-photon polymerization techniques.

7. Method according to any of the preceding claims from 4 to 6, wherein the step of realizing the plurality of electrodes (3) on the substrate provides for the sub-steps of- coating the support surface (21) of the substrate (2) with a first resist by means of coating techniques,- realizing, by depositing a first photographic mask (Ml), a spatial distribution on the support surface (21) from the resist, in the presence of a light source,- making the spatial distribution, obtained in the previous step, conductive by depositing a layer of metallic material on the support surface (21) coated by the resist,- removing the first resist from the support surface (21) of the substrate (2),- depositing a protective layer on the support surface (21) of the substrate having the metallic spatial distribution by means of thermal deposition techniques,- coating the support surface (21) having the protective layer of the previous step with a second resist by means of coating techniques,- realizing, by depositing a second photographic mask (M2), a spatial distribution on the support surface (21) coated by the second resist in the presence of the light source,- engraving the second resist at the conductive spatial distribution by means of etching techniques,- removing the second resist from the support surface (21) of the substrate (2) and rinsing the latter,8. Method for manufacturing the device (1), according to claim 7 in combination with claim 5, wherein the step of generating a map by defining the coordinates of the plurality of electrodes (3) arranged according to the predetermined spatial distribution provides for detecting the variations in the optical interface between the protective layer and the second resist.

9. Device (1) obtained by the method according to one of the preceding claims from 4 to 8, characterized in that it comprises at least a first scaffold (41) having a proliferation conformation, adapted to promote the growth of the three-dimensionalcell cluster (C), and at least a second scaffold (42), having an inhibition conformation adapted to prevent the growth of the three-dimensional cell cluster (C), wherein the proliferation conformation is a dome structure having a cavity adapted to accommodate the three-dimensional cell cluster (C) and the inhibition conformation has a tower structure.

10. System (10) for performing interferometric measurements of a three-dimensional cell cluster (C), comprising:- a device (1) according to one of the preceding claims from 1 to 3,- a stimulator (101) comprising a plurality of stimulation electrodes configured to deliver a flow of electrical current through the three-dimensional cell cluster (C),- an impedance analyser (102), in signal communication with the plurality of electrodes, said impedance analyser (102) being configured to measure the variation of electric potential between the at least one measuring electrode (31) and the at least one reference electrode (32), in the presence of the flow of electric current.

11. Method for performing interferometric measurements of a three-dimensional cell cluster (C), comprising the following steps:- providing the system (10) according to claim 10,- cultivating a three-dimensional cell cluster (C) on the support surface (21),- measuring the impedance variations, by means of the impedance analyser (102), of the three-dimensional cell cluster (C) in a predetermined time window in the presence of the flow of electric current generated by the stimulator (101).

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