Microfluidic platform suitable for cell culture, microfluidic system comprising the microfluidic platform, and uses thereof

Abstract

The present invention relates to an automated microfluidic system suitable for cell culture comprising a) a multilayer microfluidic platform suitable for cell culture comprising at least one main culture chamber and at least one corresponding secondary culture chamber, the at least one main culture chamber and the at least one corresponding secondary culture chamber are in fluid communication with each other, at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber, and at least one microvalve associated to at least one microfluidic channel, the at least one microvalve being actionable to modify, at the site of the at least one microvalve, the cross-section of the at least one microfluidic channel between an open configuration and a closed configuration; b) an automation and control system adapted to control the activation of the at least one microvalve between the open configuration and the closed configuration; and c) optionally at least one programmable pump in fluid communication with the at least one main culture chamber and / or the at least one corresponding secondary culture chamber. The present invention also relates to the aforesaid multilayer microfluidic platform. The present invention also relates to a cell culture method, comprising the steps of a) providing the automated microfluidic system; b) introducing at least one fluid in the at least one main culture chamber and / or the at least one corresponding secondary culture chamber thereof; and c) activating the automation and control system; and a method for verifying the metastatic migration of a cell line of tumour cells. The present invention also relates to the use of the microfluidic platform or the microfluidic system ( for the simulation of the metastatic process, for cell culture, for the pharmaceutical screening of active ingredients or drugs destined to the treatment of a tumour, preferably solid or liquid tumours, for the evaluation of the effects of molecules and / or nanoparticles, and / or for drug discovery.

Description

[0001] MICROFLUIDIC PLATFORM SUITABLE FOR CELL CULTURE, MICROFLUIDIC SYSTEM COMPRISING THE MICROFLUIDIC PLATFORM, AND USES THEREOF.

[0002] Field of application

[0003] The present invention refers to a microfluidic platform and microfluidic system comprising it, for cell culture, suitable for the study of intercellular communication, particularly of the metastatic process, and for the screening of drugs destined to the treatment of tumours, in particular solid or liquid tumours.

[0004] The present invention further refers to a cell culture method, a method for verifying the metastatic migration of a cell line of tumour cells, and a method for testing the influence of a substance, using the microfluidic system and / or the microfluidic platform of the invention; and the use of the microfluidic system and / or the microfluidic platform of the invention for cell culture, for the simulation of the metastatic process; for the pharmaceutical screening of active ingredients or drugs in a tumour; for the evaluation of the effects of molecules and / or nanoparticles; or for drug discovery .

[0005] In fact, the present invention finds useful use in the field of biomedicine, in particular in the field of cancer research.

[0006] Technical background

[0007] As is known, the progression of a tumour occurs in a tumour microenvironment characterized by numerous biomechanical and biochemical factors that influence the behaviour and development of the tumour and its metastases, as well as by molecules, cells and tissues that constantly interact with the tumour itself. The development of a tumour metastasis involves a complex chain of bi-directional biomechanical and biochemical signals exchanged between the metastasis itself and the main tumour site through the vascular system, which therefore also depend on the type of molecules, cells and tissues surrounding the tumour. In other words, the influence that this chain of signals has on the proliferation of the tumour and its metastasis depends on the duration over time in which the signals themselves are exchanged and the surrounding tumour microenvironment .

[0008] Such signals allow the metastatic site to actively contribute to tumour cell extravasation, migration, and colonization of the target tissue by the tumour cells. Research is increasingly developing in trying to create efficient tools to reliably investigate the nature of a cancer, its metastatic tendency (or migration) and the relationship with the surrounding tumour microenvironment, as well as to develop appropriate drug therapies and treatments.

[0009] There are currently several models for the study of tumour cells, such as conventional in vitro cell models or in vivo animal models.

[0010] In the state of the art, among conventional in vitro models, organ-on-chip systems are widely used.

[0011] Such organ-on-chip systems are microfluidic devices that make it possible to replicate in vitro the physiological functions of a human organ. In fact, such devices are micro-fabricated chips in which cell and / or tissue constructs and / or cell suspensions are seeded, which use microfluidic technology to mimic the complex characteristics of the human organs.

[0012] The organ-on-chip devices in which tumour cell cultures are grown are called tumor-on-chip. Both primary and metastatic tumour cells can be cultured within the known tumor-on-chip devices in order to test ant ineoplast ic drugs to outline a specialized therapy protocol to counteract mitotic replication of such cells, or to evaluate the tendency / metastat ic migration of a tumour. Generally, the tumor-on-chip devices comprise culture chambers or channels apt to house the cells. Furthermore, such culture chambers or channels are perfused by a liquid culture medium which represents a biomechanical stimulus for the cells cultured therein and at the same time a biochemical stimulus, thanks to the nutrients contained therein.

[0013] Generally, the microfluidic devices are made by known soft-litography techniques.

[0014] Patent application US 2023 / 0032623 Al describes a Tumor- on-a-chip device capable of replicating a tumour microenvironment in a microfluidic chip. Such a device is suitable for evaluating anticancer therapies.

[0015] Patent application CN110055176A describes a microfluidic chip that simulates a brain metastasis of a lung tumour. Patent CN109385373 describes a microfluidic co-culturing device used to detect drug sensitivity and metastatic tendency of tumours.

[0016] Patent application US 2021 / 0032584 Al describes a microfluidic chip that replicates the blood vessels for co-culture of vascular and tumour endothelial cells.

[0017] Patent application US 2018 / 0290138 Al describes a microfluidic chip for screening drug-resistant antitumour cells. Patent application US 2018 / 0172666 Al describes a microfluidic chip made of hydrogel for the co-culture of endothelial and tumour vascular cells.

[0018] However, the known microfluidic devices have some drawbacks .

[0019] The known microfluidic devices are in fact not easily adaptable and customizable with respect to the specific environmental conditions required within a study and therefore do not allow the study of cell cultures that proliferate in different tumour microenvironments. For example, the known devices do not allow the seeding of both cell monolayers and three-dimensional tumour organoids .

[0020] Furthermore, the known devices can only accommodate a rather small number of cell cultures, thus limiting the multi-organ complexity that can be achieved in the microfluidic chip.

[0021] In addition, the known devices can accommodate a single set of experimental conditions at a time, thus not being able to implement the parallel study of the main tumour and of the metastases originated by it during their development .

[0022] In addition, the known microfluidic devices do not allow to accurately replicate the change in the spatial- temporal pattern of the biomechanical and biochemical stimuli typical of a tumour microenvironment during the development of the tumour itself.

[0023] Finally, the known devices are designed to evaluate only some aspects of the complex process of metastatic dissemination and thus do not allow real-time monitoring of the process of tumour proliferation.

[0024] The object of the present invention is therefore to provide a microfluidic system and / or a microfluidic platform capable of overcoming the drawbacks of the aforesaid prior art.

[0025] It is a further object of the present invention to provide such a microfluidic system and / or microfluidic platform that is easily adaptable to the required experimental conditions.

[0026] Yet another object of the present invention is to provide such a microfluidic system and / or microfluidic platform that allows the screening and the study of drugs in order to develop personalized medicine strategies.

[0027] Summary of the invention

[0028] These and other objects are achieved according to the present invention by means of a multilayer microfluidic platform suitable for cell culture comprising:

[0029] - at least one main culture chamber and at least one corresponding secondary culture chamber, the at least one main culture chamber and the at least one corresponding secondary culture chamber being in fluid communication with each other;

[0030] - at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber; and

[0031] - at least one microvalve associated to at least one said at least one microfluidic channel, the at least one microvalve being actionable to modify, at the site of the at least one microvalve, the cross-section of the at least one microfluidic channel between an open configuration in which the area of the cross-section of the at least one microfluidic channel is maximum and a closed configuration in which the area of the crosssection of the at least one microfluidic channel is zero. The present invention further refers to an automated microfluidic system suitable for cell culture comprising : a) a multilayer microfluidic platform suitable for cell culture comprising:

[0032] - at least one main culture chamber and at least one corresponding secondary culture chamber, the at least one main culture chamber and the at least one corresponding secondary culture chamber being in fluid communication with each other;

[0033] - at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber; and

[0034] - at least one microvalve associated to at least one said at least one microfluidic channel, said at least one microvalve being actionable to modify, at the site of said at least one microvalve, the cross-section of the at least one microfluidic channel between an open configuration in which the area of the cross-section of the at least one microfluidic channel is maximum and a closed configuration in which the area of the crosssection of the at least one microfluidic channel is zero; b) an automation and control system adapted to control the activation of said at least one microvalve between said open configuration and said closed configuration; and c) optionally at least one programmable pump in fluid communication with the at least one main culture chamber and / or the at least one corresponding secondary culture chamber .

[0035] Preferably, the aforesaid microfluidic platform is automatable, i.e. it is set, and therefore suitable, to be associated to an automation and control system.

[0036] Preferably, said multilayer microfluidic platform and said automated microfluidic system are suitable for allowing the proliferation of a cell culture occupying the entire volume defined by said at least one main culture chamber and / or said at least one corresponding secondary culture chamber.

[0037] Preferably, said at least one main culture chamber and said at least one corresponding secondary culture chamber are adapted to allow, in use, the proliferation of cells throughout the volume of the chambers.

[0038] Preferably, said multilayer microfluidic platform and said automated microfluidic system are suitable for allowing a cell migration between said at least one main culture chamber and said at least one corresponding secondary culture chamber, more preferably from said at least one main culture chamber to said at least one corresponding secondary culture chamber.

[0039] Preferably, the microfluidic system comprises an incubator .

[0040] By "a corresponding secondary culture chamber" is meant a culture chamber that is in fluid communication with a main culture chamber. It is envisaged that there may be one main culture chamber, or more than one main culture chamber, each in fluid communication with its own at least one corresponding secondary culture chamber. Preferably, in case there is more than one main culture chamber, they are not in fluid communication with each other . Preferably, for each main culture chamber, in case it is provided for more than one corresponding secondary culture chamber, the corresponding secondary culture chambers are not in direct fluid communication with each other .

[0041] The expression "direct fluid communication" means a fluid communication consisting merely of a microfluidic channel or a combination of microfluidic channels.

[0042] Preferably, the at least one secondary culture chamber relative to the at least one corresponding primary culture chamber is in fluid communication with only one of said at least one corresponding main culture chamber. Preferably, secondary culture chambers relative to several main culture chambers are not in fluid communication with each other.

[0043] Preferably, said at least one main culture chamber and said at least one corresponding secondary culture chamber are structurally free, i.e. they do not contain obstructions or structural constraints to the proliferation of cells throughout the volume of the chambers .

[0044] Preferably, the at least one microvalve associated to the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber is a microvalve of the "push-up" type more preferably arranged orthogonally with respect to the microfluidic channel to which it is associated.

[0045] Preferably, the at least one microvalve is made of PDMS . Preferably, when at least two, said at least one microvalve, more preferably each of said at least one microvalve, is adapted to be activated independently of the activation of the other (s) .

[0046] Preferably, the at least one microvalve comprises a hole adapted to be associated to a respective compressed air source, more preferably with a respective compressed air generator, or with an automation and control system, for example by means of a respective needle associated to a respective tube.

[0047] Preferably, when at least two, said at least one microvalve, more preferably each of said at least one microvalve, comprises a hole adapted to be associated to a respective compressed air source, more preferably with a respective compressed air generator, or with an automation and control system, for example by means of a respective needle associated to a respective tube.

[0048] Preferably, the at least one microvalve comprises a hole associated to a respective compressed air source, more preferably with a respective compressed air generator, or with an automation and control system, for example by means of a respective needle associated to a respective tube .

[0049] Preferably, when at least two, said at least one microvalve, more preferably each of said at least one microvalve, comprises a hole associated to a respective compressed air source, more preferably with a respective compressed air generator, or with an automation and control system, for example by means of a respective needle associated to a respective tube.

[0050] In this way, it is possible to control the activation of said microvalves independently of each other.

[0051] As will be explained in more detail below, in fact, the presence of an independent connection with the compressed air source for each microvalve, either directly to the respective compressed air source (in the case of manual operation) or to the automation and control system (through a respective electrovalve) , allows the microvalves to be activated independently of each other.

[0052] As will be explained in more detail below, the crosssection of the microfluidic channel can be modified thanks to the activation of the microvalve associated to it. The microvalve may in fact be adjusted so as to be in an open configuration (of the microfluidic channel) , in which the area of the cross-section of the microfluidic channel is maximum, in a closed configuration in which the area of the cross-section of the microfluidic channel is zero, or any intermediate configuration between these two configurations, said partial closed configuration, in which the area of the cross-section of the microfluidic channel is greater than zero and less than its maximum area. In the closed configuration, the microvalve abuts against the wall of the channel thus occupying the cross-section of the microfluidic channel. This prevents the passage of fluid .

[0053] Preferably, the cross-section of the at least one microfluidic channel is semi-circular.

[0054] Preferably, the microvalve associated to a microfluidic channel defines an overlapping area (or intersection section) between the microvalve and the microfluidic channel. This area is called the microvalve area. Preferably, the area of the at least one microvalve is from 10000 pm2to 90000 pm2, more preferably from 20000 pm2to 60000 pm2, even more preferably 40000 pm2. Preferably, the area of the at least one microvalve is square, more preferably 200 pm x 200 pm in size.

[0055] Preferably, to each said at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber there is associated at least one microvalve, more preferably one microvalve, i.e. a microvalve specifically associated to said microfluidic channel.

[0056] Preferably, the number of said at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber corresponds to the number of microvalves associated to said microfluidic channels.

[0057] Preferably, the at least one main culture chamber and the at least one corresponding secondary culture chamber each comprise at least one inlet and at least one outlet. Preferably, the at least one inlet of said at least one main culture chamber is adapted for the introduction of a fluid, for example by syringe pump, in the at least one main culture chamber, and the at least one outlet of the at least one main culture chamber is adapted to remove a fluid from the at least one main culture chamber. Likewise, preferably, the at least one inlet of said corresponding secondary culture chamber is adapted for the introduction of a fluid, for example by syringe pump, in the at least one corresponding secondary culture chamber, and the at least one outlet of the corresponding secondary culture chamber is adapted to remove a fluid from the at least one corresponding secondary culture chamber .

[0058] Such at least one inlet is adapted to be associated to a pump, for example by means of respective tubes.

[0059] Preferably, said at least one inlet and / or said at least one outlet are associated, respectively, with a microfluidic channel (inlet / outlet microfluidic channels) that puts in fluid communication said at least one inlet and / or said at least one outlet with the culture chamber to which it is associated.

[0060] Preferably, the introduced fluid is chosen from a cell suspension, a fluid comprising a test substance (for example an active ingredient or a drug) , liquid culture medium, a fluid for analysis (such as for example a solution comprising antibodies) and mixtures thereof.

[0061] Preferably, the removed fluid is chosen from a cell suspension, a fluid comprising a test substance (e.g. an active ingredient or a drug) , a liquid culture medium (e.g. spent liquid culture medium) , a fluid for analysis (such as antibodies) and mixtures thereof.

[0062] Introducing a cell culture in a culture chamber is also called seeding a culture chamber.

[0063] Preferably, each main culture chamber and each corresponding secondary culture chamber comprises an inlet and an outlet.

[0064] Preferably, the pump is a syringe pump.

[0065] By the expression "control the activation", with reference to the automation and control system, it is meant herein that the automation and control system is able to switch the at least one microvalve from the open configuration to the closed configuration and vice versa. In this way, the microfluidic channel can switch from the open configuration to the closed configuration and vice versa, from the open configuration to the partially closed configuration and vice versa, from the closed configuration to the partially closed configuration and vice versa.

[0066] Preferably, the automation and control system is adapted to control the activation of at least one of said at least one microvalve, more preferably of each microvalve, between said open configuration and said closed configuration. By the expression "spent liquid culture medium" is meant herein the liquid culture medium employed in a cell culture process. Preferably, each layer of the multilayer platform is square or rectangular based, more preferably rectangular.

[0067] Preferably, the multilayer microfluidic platform has a square or rectangular, more preferably rectangular, base .

[0068] Preferably, the multilayer microfluidic platform has a maximum length from 50 to 150 mm, more preferably from 60 to 90 mm, even more preferably 75 mm; and a maximum width from 15 to 70 mm, more preferably from 30 to 50 mm, even more preferably 40 mm.

[0069] Preferably, the multilayer microfluidic platform has a maximum height from 1.5 mm to 5 mm, more preferably from 2 to 4 mm, even more preferably from 2.5 mm to 3.5 mm. The terms "height", "length" and "width" are to be understood with respect to a microfluidic platform arranged as when in use, for example resting on a surface .

[0070] The different layers of the microfluidic platform may have different dimensions. In the above paragraphs, the terms "maximum length" and "maximum width" therefore refer to that of the layer of greater size (length or width) , and "maximum height" therefore refers to the maximum height achieved by the superposition of all the layers of the microfluidic platform present.

[0071] Preferably, the automation and control system comprises:

[0072] - at least one electrovalve in fluid communication with the at least one microvalve, adapted to activate the at least one microvalve;

[0073] - a pressure regulator in fluid communication with the at least one electrovalve; and

[0074] - a computing and control unit in data communication with the at least one electrovalve and the pressure regulator .

[0075] Preferably, the pressure regulator is associated to an air generator.

[0076] Preferably, the air generator is a device adapted to generate a flow of compressed air, more preferably it is a compressor.

[0077] The term "pressure" means the pressure relative to the vacuum .

[0078] Preferably, the electrovalve is adapted to vary the pressure of said flow of compressed air to activate the at least one microvalve.

[0079] Preferably, when said at least one microvalve associated to the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber are at least two, the automation and control system is adapted to control the activation of said microvalves independently of each other. This is achieved for example by associating with said microvalves one electrovalve each.

[0080] Preferably, the automation and control system for each microvalve has association with one electrovalve each. It is therefore possible, for example, to provide for the closure of a microvalve and simultaneously the opening of another. In this way it is possible to allow for example the fluid communication between a main culture chamber and a corresponding secondary culture chamber and at the same time to close the fluid communication of the same main chamber with another corresponding secondary chamber. The flows towards and from each secondary chamber can then be controlled independently of those of the other secondary chambers. In fact, the microfluidic platform and the microfluidic system of the present invention are adapted to realize in each at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber, both a direct flow from the at least one main culture chamber to the corresponding at least one secondary culture chamber, and vice versa from the at least one corresponding secondary culture chamber to the corresponding at least one main culture chamber.

[0081] The expression "programmable pump" means an instrument suitable for the automatic and controlled introduction of a fluid.

[0082] By the expression "automatic and controlled introduction" it is meant herein that it is possible to set and regulate the flow rate of the fluid, for example, entering the at least one main culture chamber and / or the at least one corresponding secondary culture chamber .

[0083] Preferably, the pump is adapted to introduce a fluid in the at least one main culture chamber and / or the at least one corresponding secondary culture chamber.

[0084] Preferably, the automation and control system exploits software, such as for example software developed using

[0085] Arduino IDE 1.8.19.

[0086] Preferably, the electronic unit generates a digital signal connected to a computer, more preferably through the USB input of the latter.

[0087] Advantageously, the pressure variation of the flow of compressed air allows the at least one microvalve to switch from the open configuration to the closed configuration and vice versa, as described above, thus modifying, for example, the fluid communication between the at least one main culture chamber and the at least one corresponding secondary culture chamber.

[0088] This variation can be made with the automation system described above, or manually. In the case where it is made manually, this variation is made for example by connecting the hole of the at least one microvalve with a respective compressed air source, preferably a respective compressed air generator, through a respective tube.

[0089] Preferably, the pressure of the flow of compressed air is between 5 psi and 25 psi (0.344738 bar and 1.72369 bar) , more preferably between 10 psi and 15 psi (0.689476 bar and 1.03421 bar) .

[0090] Preferably, when the pressure of the flow of compressed air is between 5 psi and 10 psi (between 0.344738 bar and 0.689476 bar) , the at least one channel is in the open configuration.

[0091] Preferably, when the pressure of the flow of compressed air is between 10 psi and 15 psi (between 0.689476 bar and 1.03421 bar) , the at least one channel is in the partially closed configuration.

[0092] Preferably, when the pressure of the flow of compressed air is between 15 psi and 25 psi (between 1.03421 bar and 1.72369 bar) , the at least one channel is in the closed configuration.

[0093] As described above, preferably, the at least one microvalve is of the push-up type. In this case, as the pressure of the air flow increases, the volume of the at least one microvalve increases until it abuts against the opposite wall of the microfluidic channel until it occupies the entire cross-section of the at least one microfluidic channel.

[0094] By the expression "cross-section of the at least one microfluidic channel" is meant herein the cross-section of the microfluidic channel at the site of the at least one microvalve along a plane perpendicular to the rest plane of the platform when in use.

[0095] Preferably, the programmable pump is in fluid communication with the at least one main culture chamber and / or the at least one corresponding secondary culture chamber through the at least one inlet of the at least one main culture chamber and / or the at least one corresponding secondary culture chamber. In this way, a fluid (e.g. the cell suspension and / or the liquid culture medium) can be introduced by way of the programmable pump in the at least one main culture chamber and / or in the at least one corresponding secondary culture chamber through the respective inlets.

[0096] The exit of a fluid from the at least one main culture chamber and / or from the at least one corresponding secondary culture chamber through the respective outlets can take place in the absence of a pump.

[0097] Preferably, the at least one main culture chamber, the at least one corresponding secondary culture chamber, the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber and the inlet / outlet microfluidic channels have a width to height ratio 1:10 to 10:1, more preferably from 1:5 to 5:1, even more preferably from 1:3 to 3:1, most preferably from 1:2 to 2:1. This measure provides stability while allowing a correct flow of the fluids, without the need for a support pillar.

[0098] Preferably, the at least one microfluidic channel has a maximum width (or diameter of the half-circle section in a preferred embodiment) from 120 pm to 300 pm, more preferably from 150 to 250 pm, even more preferably 200 pm .

[0099] Preferably, at least one of said main culture chambers is in fluid communication with at least two, more preferably from two to ten, even more preferably from two to six, even more preferably four, of said corresponding secondary culture chambers.

[0100] Preferably, for at least one of said main culture chambers, the corresponding secondary culture chambers, when at least two, are in fluid communication with each other through the corresponding main culture chamber.

[0101] In other words, preferably, a main culture chamber is fluidically interposed between the corresponding secondary culture chambers .

[0102] Preferably, for at least one of said main culture chambers, the corresponding secondary culture chambers, when at least two, are in fluid communication with each other exclusively through the corresponding main culture chamber .

[0103] Preferably, for at least one of said main culture chambers, the corresponding secondary culture chambers, when at least two, are arranged around said main culture chamber .

[0104] Preferably, for at least one of said main culture chambers, the corresponding secondary culture chambers, when at least two, are on a circumference having as its centre the centre of the corresponding main culture chamber or on several concentric circumferences having as their centre the centre of the corresponding main culture chamber.

[0105] The expression "the corresponding secondary culture chambers . . . are on a circumference" means that the centre of each corresponding secondary culture chamber is on the circumference.

[0106] Preferably, at least one of said main culture chambers is at the same distance from each corresponding secondary culture chamber. In other words, preferably, the secondary culture chambers, when at least two, are on the circumference having as its centre the centre of the corresponding main culture chamber.

[0107] The latter configuration, compared to a configuration in which the corresponding secondary culture chambers are at different distances from the corresponding main culture chamber, guarantees a more balanced and symmetrical flow, improving the distribution of the gradients and reducing non-uniform shear phenomena. In addition, it facilitates access and integration of the secondary culture chambers, simplifying fluidic connection and comparative analysis between different conditions. In practical terms, this translates into greater reproducibility and experimental reliability.

[0108] Preferably, for at least one of said main culture chambers, the corresponding secondary culture chambers, when more than two, are arranged in a substantially circular shape around the corresponding main culture chamber .

[0109] Preferably, the minimum space between the secondary culture chambers is 3 mm, more preferably 2 mm.

[0110] Preferably, for at least one of said main culture chambers, the corresponding secondary culture chambers are arranged around said main culture chamber in such a way that the space between each secondary culture chamber and the two secondary culture chambers adjacent thereto is the same. In other words, preferably, the corresponding secondary culture chambers, when at least two, are on the circumference having as its centre the centre of the corresponding main culture chamber or more concentric circumferences having as their centre the centre of the corresponding main culture chamber, and are homogeneously distributed on it / them.

[0111] In a preferred embodiment, at least one of said main culture chambers is in fluid communication with four corresponding secondary culture chambers arranged around the main culture chamber and each at the same distance from the main culture chamber; in such embodiment, preferably, each corresponding secondary culture chamber is at the same distance from the two adjacent secondary culture chambers .

[0112] In a preferred embodiment, at least one of said main culture chambers is surrounded by four corresponding secondary culture chambers, which are at the vertices of a square whose diagonals intersect at the centre of the main culture chamber.

[0113] The expression "corresponding secondary chambers , which are at the vertices of a square" means that the centre of each corresponding secondary culture chamber is at the vertices of a square.

[0114] Preferably, the microfluidic platform has one or two main culture chambers, more preferably one main culture chamber .

[0115] In a preferred embodiment, the microfluidic platform comprises a main culture chamber surrounded by four corresponding secondary chambers, which are at the vertices of a square whose diagonals intersect at the centre of the main culture chamber.

[0116] However, it is possible that, when more than one, the corresponding secondary culture chambers are arranged around one or more main culture chambers at varying distances from each other, and at varying distances from the one or more main culture chambers .

[0117] Preferably, the at least one main culture chamber has a larger sectional area than the at least one corresponding secondary culture chamber.

[0118] Preferably, the at least one main culture chamber is circular in section, more preferably having a diameter from 1 to 5, more preferably from 2 to 3 mm, even more preferably 2.5 mm.

[0119] The term "section" means herein the section along a plane parallel to the rest plane of the platform when in use. Preferably, the at least one corresponding secondary culture chamber is circular in section, more preferably having a diameter from 0.5 to 3, more preferably from 1 to 2 mm, even more preferably 1.5 mm.

[0120] Preferably, the at least one main culture chamber and the at least one corresponding secondary culture chamber are substantially cylindrical in shape. Preferably, each of said at least one main culture chamber has the same dimensions and shape.

[0121] Preferably, for each main culture chamber, each of the at least one corresponding secondary culture chamber has the same dimensions and shape.

[0122] As known in the sector, "multilayer microfluidic platform" means that the platform is obtained by assembling at least two superimposed layers along an axis of development perpendicular to the resting plane of the platform when in use, such as for example a table. As will be explained in more detail below, the microfluidic platform of the invention is obtained by way of techniques known in the sector, in particular by way of microfabrication techniques.

[0123] The superimposition of the layers creates at least two levels of the microfluidic platform, as defined below. Preferably, the microfluidic platform comprises a first level comprising the at least one main culture chamber, the at least one corresponding secondary culture chamber, and the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber; and a second level comprising the at least one microvalve. The first level and the second level are described with respect to the aforesaid axis of development perpendicular to the rest plane of the platform and are substantially parallel to the resting plane.

[0124] Preferably, the first level further comprises the inlet / outlet microfluidic channels.

[0125] Preferably, the first level is above the second level, with respect to the aforesaid axis of development perpendicular to the rest plane of the platform. In this embodiment, therefore, each microvalve is below the microfluidic channel to which it is associated.

[0126] Preferably, the microfluidic platform is obtained by assembling a first layer, a second layer and a third layer .

[0127] Preferably, the first layer and the second layer are each a formed layer of PDMS obtained by microfabrication techniques .

[0128] Other materials that may be used are polymethylmethacrylate (PMMA) , polyacrylates, polycarbonates, polycyclic olefins, polyimides, and polyurethanes .

[0129] Preferably, the third layer is a sheet of solid and chemically inert, transparent material sterilizable for example in an autoclave, more preferably glass.

[0130] Preferably, the first layer is coupled to the second layer, and the third layer is coupled to the second layer .

[0131] Preferably, a fourth layer is interposed between the second layer and the third layer. Said fourth layer is preferably a PDMS layer.

[0132] Preferably, the interface between the first layer and the second layer determines the at least one main culture chamber, the at least one corresponding secondary culture chamber, and the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber, and the interface between the third layer or the fourth layer and the second layer determines the at least one microvalve associated to said at least one microfluidic channel .

[0133] Preferably, the interface between the first layer and the second layer further determines the inlet / outlet microfluidic channels. As is evident to the person skilled in the art, this configuration ensures that the at least one microvalve is not on the same plane, when in the open configuration, with the at least one microfluidic channel, to which it is associated.

[0134] Preferably, the microfluidic channel is not in fluid communication with the at least one microvalve.

[0135] Preferably, the first layer and the second layer are rectangular in section.

[0136] The present invention also refers to a cell culture method, comprising the steps of: a) providing the automated microfluidic system comprising the microfluidic platform of the invention; b) introducing at least one fluid in the at least one main culture chamber and / or the at least one corresponding secondary culture chamber; and c) activating the automation and control system, preferably by way of a suitable set program.

[0137] Preferably, the aforesaid steps are in sequence. Preferably, step b) is preceded by a step a2) , wherein the at least one microvalve associated to the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber of the microfluidic platform is set in a closed configuration .

[0138] Preferably, step b) is performed by means of said programmable pump. Preferably, for at least one main culture chamber, all the aforesaid microvalves of the microfluidic platform are set in closed configuration.

[0139] Preferably, in step b) the fluid is chosen from cell suspension, liquid culture medium, and mixtures and combinations thereof, more preferably cell suspension. Preferably, step b) comprises introducing at least one fluid in the at least one main culture chamber and at least one fluid in the at least one corresponding secondary culture chamber.

[0140] Preferably, the cell suspension comprises cells chosen from tumour cells and cells representing a target metastatic site of a tumour cell.

[0141] Preferably, the cell suspension comprising tumour cells comprises a cell line chosen from cell line of neuroblastoma cells, cell line of glioblastoma cells, cell line of rectal colon tumour cells, and cell line of ovarian tumour cells, more preferably cell line of neuroblastoma cells, even more preferably cell line of neuroblastoma SK-N-AS cells.

[0142] Preferably, the cells representing a target metastatic site of a tumour cell are chosen from cell line of stem cells and cell line of endothelial cells.

[0143] Preferably, the cell suspension comprising tumour cells comprises the cell line of neuroblastoma SK-N-AS cells. Preferably, the cell suspension comprising cells representing a target metastatic site of a tumour cell comprises a cell line chosen from cell line of human mesenchinal stem cells (hMSC) and cell line of human umbilical vein endothelial cells (HUVEC) . Preferably, step b) comprises introducing in the at least one main culture chamber a cell line of tumour cells, more preferably chosen from cell line of neuroblastoma cells, cell line of glioblastoma cells, cell line of rectal colon tumour cells, and cell line of ovarian tumour cells, even more preferably cell line of neuroblastoma cells, even more preferably cell line of neuroblastoma SK-N-AS cells.

[0144] Preferably, step b) comprises introducing in the at least one corresponding secondary culture chamber a cell line representing a target metastatic site of a tumour cell, more preferably chosen from cell line of human mesenchinal stem cells (hMSC) and cell line of human umbilical vein endothelial cells (HUVEC) .

[0145] For at least one main culture chamber, the corresponding secondary culture chambers, when at least two, can be seeded with a cell suspension of the same type or with cell suspensions of a different type from each other. Preferably, in step b) the flow of fluid in the at least one inlet / outlet microfluidic channel is from 0.1 to 20 pl / min, more preferably from 0.5 to 10 pl / min, even more preferably 2 pl / min.

[0146] Preferably, in step b) the cell suspension is introduced so as to obtain a final density from 50 to 500 cells / mm2, more preferably from 70 to 300 cells / mm2, even more preferably from 80 to 250 cells / mm2of chamber bottom surface .

[0147] Preferably, the final density of cells in the at least one main culture chamber is greater than that of the at least one corresponding secondary culture chamber. Preferably, the final density of SK-N-AS is 200 cells / mm2; the final density of hMSCs is 100 cells / mm2; and the final density of HUVEC is 150 cells / mm2. Preferably, step c) is preceded by a resting step bl) , more preferably at a temperature of 37°C; for a time of from 0.5 to 12 hours, more preferably from 1 to 4 hours. Preferably, the resting step bl) is performed in an incubator. In this step, preferably the at least one microvalve is closed.

[0148] Steps a) to b) correspond to seeding the platform. Preferably, step c) comprises the entry of a flow of fluid, more preferably liquid culture medium, in the at least one main culture chamber and / or in the at least one corresponding secondary culture chamber; and / or removing a flow of fluid from the at least one main culture chamber and / or from the at least one corresponding secondary culture chamber. Step c) corresponds to the cell culture.

[0149] Such flow entry and / or removal may be continuous or programmed so as to be performed at predetermined intervals .

[0150] Preferably, step c) comprises activating the at least one microvalve associated to the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber and the programmable pump according to the set program. Preferably, in step c) , activating the at least one microvalve associated to the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber comprises putting the at least one microvalve in an open configuration .

[0151] Preferably, in step c) the flow of fluid in the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber is from 0.1 to 10 pl / min, more preferably from 0.5 to 5 pl / min, even more preferably 1 pl / min.

[0152] Preferably, the pressure applied is from 8 to 17 psi, more preferably from 10 to 15 psi, even more preferably 12 psi .

[0153] Preferably, step c) is performed for a time from 2 hours to 2 weeks, more preferably from 3 days to 10 days, most preferably 7 days.

[0154] In a preferred embodiment, neuroblastoma SK-N-AS is introduced into the at least one main culture chamber while hMSCs and / or HUVEC are introduced into the at least one corresponding secondary culture chamber.

[0155] The present invention also refers to a method for verifying metastatic migration of a cell line of tumour cells, comprising the steps of: a) providing the automated microfluidic system comprising the multilayer microfluidic platform of the invention; b) introducing at least one fluid in the at least one main culture chamber and / or the at least one corresponding secondary culture chamber; and c) activating the automation and control system, preferably by way of a suitable set program; wherein in step b) a cell line of tumour cells is introduced in the at least one main culture chamber and a cell line representing a target metastatic site of the cell line of tumour cells is introduced in the at least one secondary culture chamber; and d) detecting the possible presence of metastasis of the cell line of tumour cells in the at least one corresponding secondary chamber.

[0156] Preferably, the aforesaid steps are in sequence.

[0157] Preferably, step b) is preceded by a step a2) , wherein the at least one microvalve associated to the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber of the microfluidic platform is set in a closed configuration .

[0158] Preferably, step b) is performed by means of said programmable pump.

[0159] Preferably, for at least one main culture chamber, all the aforesaid microvalves of the microfluidic platform are set in closed configuration.

[0160] Preferably, steps a) to c) are performed according to the preferred features described above in relation to the cell culture method.

[0161] The expression "detecting the possible presence of metastasis of the cell line of tumour cells" in step d) means the detection of the presence of cells belonging to the aforesaid cell line of tumour cells.

[0162] In particular, the detected cells belonging to the aforesaid cell line of tumour cells are from the at least one corresponding primary culture chamber.

[0163] Following the opening of the at least one microvalve associated to the at least one microfluidic channel adapted to put in fluid communication the at least one main culture chamber with the at least one corresponding secondary culture chamber, in fact, cells belonging to the aforesaid cell line of tumour cells can spontaneously migrate from the at least one main culture chamber to the at least one corresponding secondary culture chamber. Preferably, in fact, said metastatic migration is a spontaneous metastatic migration.

[0164] Preferably, step d) is performed by way of a fluorescence staining technique, more preferably chosen from a viability assay, PCR and immunofluorescence.

[0165] Preferably, the fluorescence staining is performed with the use of a fluorescent marker, more preferably chosen from Hoechst, calcein-AM, tet ramethylrhodamine- conjugated phalloidin (TRITC) , 4 ' , 6-diamidine-2- phenylindole (DAPI) , ant i-Viment in monoclonal antibody, Anti-E cadherin monoclonal antibody, HIF-1 alpha monoclonal antibody, Anti-NANOG monoclonal antibody, Anti-SOX2 antibody, Alexa Fluor 488 conjugated antibodies, Alexa Fluor 594 conjugated antibodies, Alexa Fluor 647 conjugated antibodies, propidium iodide, and combinations thereof.

[0166] Preferably, the viability assay is the Live&Dead assay, which comprises the use of Hoechst fluorescent markers and calcein-AM.

[0167] Preferably, the immunofluorescence technique comprises the use of the primary antibody Vimentin.

[0168] Preferably, the immunofluorescence technique comprises the use of the Goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488. Preferably, the immonof luorescence technique comprises the measurement of the mean fluorescence intensity (MFI) by means of known image analysis techniques, implemented for example by means of a confocal fluorescence microscope and subsequent image processing by means of

[0169] Image J software.

[0170] Preferably, the MFI measurement is performed on the cell line of tumour cells in the at least one corresponding secondary culture chamber.

[0171] The present invention also refers to a method for testing the influence of a test substance adapted to induce a cellular response, preferably a drug, on a cell culture comprising performing a cell culture method according to the invention, wherein step c) comprises putting in contact the cell culture with the test substance, preferably an active ingredient of the drug or active ingredient, incubating in controlled (environmental and spatial-temporal) conditions, and evaluating the effect of the substance, more preferably by way of a fluorescence staining technique.

[0172] The present invention also refers to the use of the microfluidic platform or the automated microfluidic system of the invention for cell culture, for the simulation of the metastatic process; for the pharmaceutical screening of active ingredients or drugs destined to the treatment of tumours, preferably solid or liquid tumours; for the evaluation of the effects of molecules and / or nanoparticles; and / or for drug discovery .

[0173] Brief Description of the Figures

[0174] Figure 1 shows a schematic representation (not to scale) of the microfluidic system according to a preferred embodiment of the present invention wherein the microfluidic platform is shown in a top plan view.

[0175] Figure 2 shows two photographs (2a, 2b) of the microfluidic platform according to a preferred embodiment of the present invention in a top view. Figure 2c shows a detail of the microfluidic platform, in particular a microfluidic channel between a main chamber and a corresponding secondary chamber and the microvalve associated thereto.

[0176] Figure 3 shows a schematic representation (not to scale) of the microfluidic platform according to two preferred embodiments (3a and 3b) of the present invention wherein the microfluidic platform is shown in a front view (i.e. , the length can be seen) .

[0177] Figures 4a and 4b show a schematic representation (not to scale) of the microfluidic platform according to two preferred embodiments of the present invention wherein the microfluidic platforms are shown in a perspective view .

[0178] Figures 4c and 4d show schematically (not to scale) a platform according to a preferred embodiment for activating the open and closed configuration of the microfluidic channel respectively by means of the microvalve .

[0179] Figure 5 (a-f) shows various sequential time points of the validation experiment of Example 3: a) closed valve (P>0) ; b) open valve (P = 0)t = 0s; c) open valve (P=0)t=3s; d) open valve (P=0)t=10s; e) open valve (P = 0)t = 15s; f) closed valve (P>0) . Figures 5cc and 5ff represent details of Figures 5c and 5f, respectively. P = pressure.

[0180] Figure 6 shows the staining of SK-N-AS cells 48 hours after seeding in the multilayer platform of the invention (4X and 10X magnification) according to the experiment of Example 4.2: in the images in the middle (Hoechst) the cell nuclei stained with HOECHST are shown; in the images on the left (Calcein-AM) the cell cytoplasm of the only still viable cell population stained with calcein is shown; and in the images on the right (Merge) the overlay of the two previous images is shown. Scale bar 1000 and 400 pm.

[0181] Figure 7 shows the staining of hMSC cells 48 hours after seeding in the multilayer platform of the invention (4X and 10X magnification) according to the experiment of Example 4.2: in the images in the middle (Hoechst) the cell nuclei stained with HOECHST are shown; in the images on the left (Calcein-AM) the cell cytoplasm stained with calcein is shown and in the images on the left (Merge) the overlay of the two previous images is shown. Scale bar 1000 and 400 pm.

[0182] Figure 8 shows the staining of HUVEC cells 48 hours after seeding in the multilayer platform of the invention (4X and 10X magnification) according to the experiment of Example 4.2: in the images in the middle (Hoechst) the cell nuclei stained with HOECHST are shown; in the images on the left (Calcein-AM) the cell cytoplasm stained with calcein is shown and in the images on the right (Merge) the overlay of the two previous images is shown. Scale bar 1000 and 400 pm.

[0183] Figure 9 shows a scheme of the co-culture experiment of Example 4.3: (a) SK-N-AS and hMSC experiment; (b) SK-N- AS and HUVEC experiment .

[0184] Figure 10 shows the staining of SK-N-AS cells 72 hours after seeding the main culture chamber according to Example 4.3: the cell nuclei are stained with DAPI and F-actin is stained with Phalloidin and the overlay is shown in the images below ("Merge") . Scale bar 1000, 400 and 200 pm.

[0185] Figure 11 shows the staining of SK-N-AS and hMSC cells after 72 hours from seeding in the secondary culture chambers according to Example 4.3: the cell nuclei are stained with DAPI and F-actin is stained with Phalloidin and in the images below ("Merge") the overlay is shown. Scale bar 1000, 400 and 200 gm.

[0186] Figure 12 shows the staining of SK-N-AS and HUVEC cells after 72 hours from seeding in the secondary culture chambers according to Example 4.3: the cell nuclei are stained with DAPI and F-actin is stained with Phalloidin and in the images below (Merge) the overlay is shown. Scale bar 1000, 400 and 200 gm.

[0187] Figure 13 shows the IF staining results of Example 4.3. 20x magnification for SK-N-AS and hMSCs co-cultured in the microfluidic platform 72 hours after seeding. The cell nuclei are stained with DAPI, while Vimentin is stained with Alexa Fluor 488. The overlay is shown in the images on the right (Merge) . Scale bar 50 gm.

[0188] Figure 14 shows the IF staining results of Example 4 for Vimentin protein in SK-N-AS. Representative images for the expression of Vimentin in SK-N-AS after 72 hours from seeding in both tests. The cell nuclei are stained with DAPI, while Vimentin is stained with Alexa Fluor 488. The overlay is shown in the images on the right (Merge) . Scale bar 50 gm.

[0189] Figure 15 shows the IF staining results of Example 4 for Vimentin protein in hMSCs. Representative images for the expression of Vimentin in hMSCs after 72 hours from seeding in both tests. The cell nuclei are stained with DAPI, while Vimentin is stained with Alexa Fluor 488. The overlay is shown in the images on the right (Merge) . Scale bar 50 gm.

[0190] Figure 16 shows the results for calculation of the mean fluorescence intensity (MFI) of Vimentin for SK-N-AS. (a) Boxplot for calculation of MFI for SK-N-AS test cells. The box plots represent values from at least n=3 independent experiments. *p<0.05, **p<0.01 and ** *p<0.001 vs control; (b) column diagram for MFI analysis results. All values are means ± SEM from at least three independent experiments.

[0191] Figure 17 shows the results for the calculation of MFI of Vimentin. (a) Boxplot for calculation of MFI for hMSC test cells. The box plots represent values from at least n=3 independent experiments. *p<0.05, **p<0.01 and ** *p<0.001 vs control; (b) column diagram for MFI analysis results. All values are means ± SEM from at least three independent experiments.

[0192] Detailed description of the invention

[0193] The features and advantages of the microfluidic system according to the present invention will be more apparent from the following description, which is to be understood as exemplifying and not limiting, with reference to the appended schematic drawings.

[0194] With reference to the figures, in particular Figure 1, according to a preferred embodiment of the present invention, the microfluidic system, overall indicated with 1, comprises a multilayer microfluidic platform 2 which in turn comprises a main culture chamber 3 (which is shown here in circular section, but which can be of any other shape) , four corresponding secondary culture chambers 4 (which are shown here in circular section, but which can be of any other shape) , four microfluidic channels 5 which put in fluid communication the main culture chamber 3 with each of the corresponding secondary culture chambers 4. The secondary culture chambers 4 are placed at the same distance from the main culture chamber 3, which is at their centre. The secondary chambers 4, according to this preferred embodiment, are positioned at the vertices of a square whose diagonals intersect at the centre of the main culture chamber 3. Therefore, each of the four secondary chambers 4 is at the same distance from the adjacent secondary chambers on both sides.

[0195] The main culture chamber 3 has an inlet 6 and an outlet 7. Likewise, each of the corresponding secondary culture chambers has an inlet 8 and an outlet 9 each. These inlets and outlets are connected to the culture chamber associated to them through a microfluidic channel (inlet / outlet microfluidic channel) .

[0196] Finally, each microfluidic channel 5 has a microvalve 10 positioned along the length of the microfluidic channel. The microvalve 10 may be positioned at any position along the microfluidic channel 5. It is possible that one or more microfluidic channels 5 are not associated to a microvalve 10, but it is preferred that each channel has a microvalve associated to it.

[0197] The number of main culture chambers 3 and corresponding secondary culture chambers 4 is variable, as is their positioning, and may depend on the specific use one wishes to make of the microfluidic system 1.

[0198] As mentioned above, the microfluidic platform 2 may have only one main culture chamber 3 and corresponding secondary culture chambers 4, which may be in any number, or may have several main culture chambers 3 each in fluid communication with their own corresponding secondary culture chambers 4. The presence of more than one main chamber 3, each in fluid communication with its own corresponding secondary culture chambers 4, allows, for example, to perform several tests in parallel.

[0199] Preferably, in such an embodiment, the different main culture chambers 3 are not in fluid communication with each other. Preferably, the corresponding secondary culture chambers relative to different main culture chambers are not in fluid communication with each other. Preferably, for each main culture chamber 3, in case it provides for at least two corresponding secondary culture chambers 4, the corresponding secondary culture chambers 4 are not in direct fluid communication with each other. In fact, according to preferred embodiments of the present invention, the secondary culture chambers 4 relative to a main culture chamber 3 are in fluid communication with each other through the corresponding main culture chamber 3, preferably exclusively through the corresponding main culture chamber 3.

[0200] Figure 1 also shows the automation and control system 11 connected to the platform at the microvalves 10, more in particular the hole 12 of the microvalves (the figure shows only one connection to a single microvalve) by means of the respective tubes.

[0201] Figure 1 also shows a programmable pump 13 connected to the platform at the inlets 6, 8 (only one connection to an inlet is shown) through respective tubes.

[0202] In Figure 1, the section of the main culture chamber 3 is larger than that of each of the corresponding secondary culture chambers 4, which in turn have identical dimensions to each other. However, it is possible that the chambers are all in the same size between them, or that the secondary culture chambers 4 are different in size and occupy different areas from each other. Preferably, said at least one main culture chamber 3 and said at least one corresponding secondary culture chamber 4 are structurally free, i.e. they do not contain obstructions or structural constraints, such as for example pillar, in order to allow cell proliferation throughout the whole volume defined by the perimeter of the chambers .

[0203] Figure 2 shows photos from above of the platform in which the elements of the platform schematically illustrated in figure 1 can be seen.

[0204] In particular, the inlets 6, 8 and the outlets 7, 9 of the main chamber 3 and the secondary chambers 4, respectively, are clearly visible. It can also be found that said inlets 6, 8 and outlets 7, 9 are suitable to be associated for example with tubes for carrying out the introduction of fluid into the chambers, for example cell suspension, a fluid comprising a test substance (for example an active ingredient or a drug) , a liquid culture medium (for example liquid culture medium) , a fluid for analysis (such as for example antibodies) , for example by means of a syringe pump, and for the removal of a fluid.

[0205] Figure 2c clearly shows the area where the microfluidic channel 5 overlaps the microvalve 10. As will be seen more clearly in Figure 4, the channel 5 and the microvalve 10 lie on two distinct planes, and are not in fluid communication with each other.

[0206] The area where the channel 5 and the microvalve 10 overlap is defined as the area of the microvalve. In a preferred embodiment it is 200 pm x 200 pm, but of course it may vary according to needs.

[0207] Preferably, the at least one inlet 6.8 and the at least one outlet 7.9 have a diameter from 0.5 to 1.5 mm, more preferably from 0.8 to 1.2 mm, even more preferably 1 mm .

[0208] Preferably, each microfluidic channel 5 is associated to a microvalve 10, i.e. a microvalve 10 specifically associated thereto.

[0209] Preferably, each main culture chamber and each corresponding secondary culture chamber has an inlet 6, 8 and an outlet 7, 9.

[0210] Figure 3a shows a schematic representation of a preferred embodiment of the microfluidic platform of the invention having geometry corresponding to that of Figure 1. It can be seen that in this figure, as in figure 3b and Figure 4, the platform has been cut so that the geometry is rotated by 45° with respect to the representation of Figure 1. This difference does not affect the operation of the microfluidic platform or microfluidic system. From the figure it can be seen that the microfluidic platform is a multilayer platform and is made on three layers A, B, C, of which the layer C is placed at the lower level, i.e. on the surface on which it is rested in use (for example a table) , while the layer B is above the layer C, and the layer A on the layer B.

[0211] Figure 3b shows a schematic representation of an alternative preferred embodiment of the microfluidic platform of the invention having geometry corresponding to that of Figure 1. From the figure it can be seen that the microfluidic platform is a multilayer platform and is made on four layers A, B, C, and D of which the layer C is placed at the lower level, i.e. on the surface on which it is rested in use (for example a table) , while the layer D is above the layer C, the layer B is above the layer D, and the layer A on the layer B.

[0212] In both embodiments 3a and 3b, the layer A and the layer B are obtained by way of microfabrication techniques known to the person skilled in the art and are preferably made of polydimethylsiloxane (PDMS) . The layer D is a PDMS layer. Other materials suitable for the purpose (for the layers A, B and D) may be used, such as for example polymethylmethacrylate (PMMA) , polyacrylates, polycarbonates, polycyclic olefins, polyimides, and polyurethanes. The layer C, in the preferred embodiment, is made of glass.

[0213] The main chamber 3, the secondary chambers 4, the microfluidic channels 5 and the inlet / outlet microfluidic channels are on the same first level (first level) and are made in and defined by the interface between the layers A and B (since these are both represented with the same colour, they are not distinguishable in Figure 4) . The microvalves are instead made in the and defined by the interface between the layers C or D, and B and are on a second level.

[0214] The at least one microvalve 10, when in open configuration, lies on a different plane, which may be above or below the plane of the at least one microfluidic channel 5.

[0215] Preferably, the microvalve 10 never enters into fluid communication with the at least one main culture chamber 3 and / or the at least one secondary culture chamber 4 and / or the at least one microfluidic channel 5 of the microfluidic system, so as not to be contaminated with the fluids present in the at least one main culture chamber 3 and / or in the at least one secondary culture chamber 4 and / or in the at least one microfluidic channel 5. This measure allows the microvalve to be kept in optimal conditions for a long time of activity of the microfluidic system.

[0216] The dimensions of the microfluidic platform and its parts are dictated by practical needs known to the person skilled in the art. The minimum space between structures (excluding the channels) must be 2 mm. Furthermore, as described above, preferably, the main and secondary culture chambers, and the microfluidic channels have a width to height ratio from 1:10 to 10:1. This measure provides stability while allowing a correct flow of the fluids, without the need for, for example, a support pillar .

[0217] The production technique of the platform is based on the production of layers of, for example, PDMS obtained starting from moulds made with positive or negative photoresist .

[0218] Preferably, the moulds are obtained by way of known soft lithography techniques.

[0219] In the preferred embodiment, the layer A, so-called "flow layer", is obtained by replica moulding starting from a mould obtained with the deposition of positive photoresist and, subsequently, negative photoresist superimposed. The layer B, so-called "control layer", is obtained by replica moulding starting from a mould obtained with the deposition of negative photoresist. The mould of the (flow) layer A is therefore composed of two different photoresists as explained above, one positive and one negative. The negative photoresist is used to form a part (in particular, the upper part) of the outline of the culture chambers, while the positive photoresist to form a part (in particular, always the upper part) of the outline of the microfluidic channel, said outlines both being made on the same mould of the flow layer. Furthermore, in a preferred embodiment, for the production of the mould of the (flow) layer A and in particular for the production of the outline of the microfluidic channel and of the culture chambers, two photomasks are necessary, one to be superimposed on the positive photoresist layer, and one to be superimposed on the negative photoresist layer.

[0220] The mould of the (control) layer B, on the other hand, is composed of negative photoresist which is used to form the remaining part of the outline of the culture chambers, the microfluidic channel and the microvalve (i.e. , the lower part) . Furthermore, in a preferred embodiment, for the production of the control layer mould and in particular for the production of the remaining part of the outline of the culture chambers, of the microfluidic channel and of the microvalve, a single photomask is necessary to be superimposed on the negative photoresist layer.

[0221] In other words, since the microfluidic channels 5 of the platform are defined by the interface between the layer A and the layer B, on one side, such channels are manufactured starting from the positive photoresist mould (flow layer mould) and on the other side starting from the negative photoresist mould (control layer mould) . Furthermore, since the culture chambers 3, 4, are defined by the interface between the layer A and the layer B, on one side, such chambers are manufactured starting from the negative photoresist mould (flow layer mould) and on the other side starting from the negative photoresist mould (control layer mould) . On the other hand, the production of a microvalve 10 involves the use of two photoresists: one preferably positive for the flow layer mould and the other preferably negative for the control layer mould. The orthogonal intersection section between the control layer and the region of the flow layer coupled to it forms the push-up valve. In figure 4c it can be seen that the microfluidic channel 5 is in the open position. The semicircular section of the channel is in fact unobstructed. Conversely, in figure 4d) the wall of the microvalve is adjacent to the wall of the microfluidic channel thus obstructing the passage of fluid in the microfluidic channel (to facilitate the understanding of figures 4c) and 4d) , a part of the microvalve has been highlighted. Regarding the flow layer A mould, the positive photoresist is preferred in order to give roundness to the microfluidic channels, i.e. to confer the preferred semicircular cross-section, to optimize the closure of the microvalve. However the other components of the flow geometry (culture chambers) are manufactured starting from mould with negative photoresist (both the flow layer and control mould) as the positive resistors tend to be harder after heat treatment and generate a high flow resistance.

[0222] As explained, each culture chamber 3, 4 comprises an inlet 6, 8 and an outlet 7, 9, for example respectively for cell seeding, perfusion of liquid culture medium and discharge of the spent liquid culture medium. To ensure a proper operation of the microfluidic valves, their area (calculated as the product between the width of the microfluidic channel and the width of the microvalve) must be minimized. The width of the control channel is 200 |im, hence the area of the microvalve is 200 |im x 200 |lm (the minimum area for proper closing of the microvalve must be at least 150 x 150 |im) .

[0223] Figure 2 shows the assembled PDMS microfluidic platform after replica moulding and plasma treatment. The PDMS structure is attached to a 75x50 cm slide. Figure 2 (c) highlights the presence of two levels in the assembled platform: the culture chambers in the first level comprising the culture chambers and the microfluidic channels, and in a different colour the second level below, which comprises the microvalve.

[0224] As for the automation and control system, it has the function of controlling the flow of fluid both inside the culture chambers and in the microfluidic channels. For this purpose, the programmable pump is connected with the inlets 6, 8 thus controlling the flow of fluid in input to the chambers. As for the flow of fluid in the microfluidic channels, which connect the main chamber and the secondary chambers, it is further controlled by the electrovalve, which activates the microvalves between two configurations, open and closed one. By adjusting the pump and the electrovalve, the flow of fluid within the platform can be controlled and regulated over time. Preferably, all the microvalves are driven by electrovalves which are in turn controlled by an electronic unit for the generation of the digital signal (Arduino Uno Rev3 SMD) connected to the USB port of a computer. Preferably, each electrovalve can change the microvalves from atmospheric pressure (open position) to the pressure that allows the microvalves to be closed through compressed air

[0225] As explained above, it is possible to control the activation of the microvalves independently of each other. This is achieved for example by associating with said microvalves one electrovalve each.

[0226] Arduino is open-source electronics prototyping platforms based on flexible, easy-to-use hardware and software. The Arduino boards can read different inputs and convert them into outputs that are capable of activating the micro components integrated into the PDMS platform. Arduino hardware control can be performed using the Arduino programming language through Arduino Software (IDE) or Matlab®. An appropriate Arduino code must be developed based on the specific application.

[0227] In a preferred embodiment, each platform has four microvalves 10 that are independently controlled to control fluid communication between each of the four corresponding secondary culture chambers and the main culture chamber. According to this preferred embodiment, the automation system has four independent electrovalves to allow the connection of the microfluidic platform or eight independent electrovalves to allow the connection of two microfluidic platforms at the same time. The Arduino hardware can also be controlled using an appropriate Matlab® script which, unlike Arduino, allows to create a user-oriented interface to facilitate valve control. At this point, four tubes connected to the electrovalves are fixed in the holes of the control layer on the platform, at the inlets of the microvalves.

[0228] Even in the absence of an automation system, it is possible to ensure the independence of the microvalves by connecting them, for example, each (or more than one) directly to a compressed air source, such as a compressed air generator, for example through a needle associated to a tube, the activation of each microvalve being manually operable.

[0229] When in use, e.g. for performing a cell culture, the at least one main culture chamber and the at least one corresponding secondary culture chamber will be seeded e.g. with the respective cell lines suspended in their specific liquid culture medium for their growth. This operation will be carried out when the microvalves 10 are in the closed configuration, to prevent the contents of the two chambers from migrating, that is, from migrating from the main chamber to a corresponding secondary chamber and / or vice versa. The platform is then allowed at an incubation temperature for a predetermined time so that cell cultures can proliferate. Once the chambers are ready, the automation and control system can be activated.

[0230] It is then possible, for example, to activate the microvalves 10 in open configuration so as to put in communication each main culture chamber with each corresponding secondary culture chamber.

[0231] According to a preferred embodiment, each main chamber is seeded with neuroblastoma cells, and each corresponding secondary chamber is seeded with target cells such as for example HUVEC or hMSC.

[0232] The operator can then program the automation and control system so as to mimic the cellular environment in vivo and observe the behaviour of the neuroblastoma cells, in particular their migration towards the target metastatic cells .

[0233] The ex vivo model made by the microfluidic system of the invention allows not only to study and observe the progression of a cancer and the formation of metastases but also to test potential anticancer drugs.

[0234] EXAMPLE 1

[0235] Manufacture of the microfluidic platform

[0236] 1.1. Soft lithography for the mould part for the flow layer with positive photoresist

[0237] The photomask was obtained using AutoCad software (manufactured by Autodesk, USA) . Then, the geometry (design) was imported into Adobe Illustrator (Adobe®) which allows to colour the photomask. A photomask allows to transfer a specific geometry onto a photoresist layer when exposed to a UV light source. In this case, two photomasks were used, one for the part of the mould with positive photoresist for the flow layer (A) and the other for the part of the mould with negative photoresist for the flow layer (A) .

[0238] A silicon substrate (4-inch diameter) was washed with Acetone (Sigma-Aldrich, USA) , Methanol (Sigma-Aldrich, USA) , and with distilled water to promote photoresist adhesion for all subsequent steps. The substrate was dried using compressed air and placed on a hot plate at 105 °C for at least 10 minutes in order to remove all humidity. After this pre-t reatment , the silicon substrate was treated with Hexamethyldisilazane vapour (HDMS, Sigma-Aldrich, USA) for at least 10 minutes. HDMS is a primer that acts as a adhesion promoter for photoresist by modifying the surface of the silicon substrates from hydrophilic to hydrophobic. The substrate was inserted into a spin-coater (WS-650-23NPP, Laurell, Laurell Technologies Corporation, USA) and secured to the chuck by vacuum generated by a pump. At this point a layer of positive photoresist SPR-220-7 (Rohm and Haas, Dow Corning, USA) was distributed over the substrate and spin-coated to obtain a thickness of 15-20 pm on the substrate. The spin-coating protocol was as follows :

[0239] - first step: 250 revolutions per minute for 15 seconds with an acceleration of 1064 revolutions per minute / s ;

[0240] - second step: 560 revolutions per minute for 75 seconds with an acceleration of 1064 revolutions per minute / s .

[0241] After photoresist deposition, a Soft Bake process was carried out by placing the substrate on a hot plate at 90 °C for 10 minutes. The temperature and heating time depend on the height of the photoresist layer.

[0242] After the Soft Bake treatment, the silicon substrate was again treated with Hexamethyldisilazane vapour (HDMS, Sigma-Aldrich) for 10 minutes, and then, a second layer of the same photoresist was spun over the first one. The spin-coating protocol to obtain a final thickness of 35- 40 pm was as follows:

[0243] - first step: 250 revolutions per minute for 15 seconds with an acceleration of 1064 revolutions per minute / s ;

[0244] - second step: 560 revolutions per minute for 105 seconds with an acceleration of 1064 revolutions per minute / s .

[0245] At this point, a second Soft Bake treatment was performed, with heating at 90 °C for 60 minutes. Afterwards, the substrate was kept in the dark for at least 3 hours to rehydrate the photoresist.

[0246] At this point it was necessary to transfer the photomask image onto the resist-coated substrate through the activation of the photosensitive components of the photoresist. Before the exposure step, the photomask was aligned with the substrate to polymerize the positive photoresist in the areas not exposed to UV light. A UV lamp with a wavelength of 365 nm was used for the exposure step. The parameters to be set are the exposure energy Eexpin [mJ / cm2] which depends on the thickness of the resist layer and the exposure time texp in [s] . The latter, if the lamp power P in [mW / cm2] is known, can be calculated with the following equation: texp=Eexp*P. In the present case, the exposure energy required was 2000 mJ / cm2. After exposure, the substrate was developed in a solvent composed of water (97-98%) and tet ranethylammonium hydroxide (2.45%) , (Microposit MF- 319, Microchem, CHIMIE TECH SERVICE, Milan, Italy) to dissolve the uncrosslinked polymer. The substrate was immersed in the solvent in a glass trough placed over a rocking shaker for approximately 15 minutes. The substrate was then rinsed with distilled water to stop its development and dried with compressed air. The final step was the heat treatment called Hard Bake. This step is necessary to make the remaining photoresist and the traces of remaining solvents evaporate and improve the mechanical and thermal characteristics of the mould. The Hard Bake process was performed by placing the substrate on a hot plate for 10 minutes at 65 °C and then setting a ramp of 10 °C / hour for 15 hours to reach the final temperature of 190 °C / hour.

[0247] 1.2. Soft lithography for the mould part for the flow layer with negative photoresist

[0248] After the 15 hours required for the Hard Bake process, a negative photoresist layer SU-8 2100 (Microchem) was spin-coated over the substrate (as obtained at the end of the processes set out in the previous paragraph) to obtain a thickness of 250 pm.

[0249] The spin-coating protocol for this layer was as follows:

[0250] - first step: 500 revolutions per minute for 10 seconds with an acceleration of 100 revolutions per minute / s ;

[0251] - second step: 1000 revolutions per minute for 32 seconds with an acceleration of 300 revolutions per minute / s .

[0252] The silicon substrate was left on a flat surface for at least 10 minutes at room temperature to better even out the height of the photoresist layer. The Soft Bake process was then performed by placing the substrate on a hot plate at 95 °C for 45 minutes. The ideal times are reported in Table 1, as they are dependent on the height of the photoresist layer.

[0253] Table 1 - Soft Bake process times (min) in relation to the thickness (pm) of the photoresist SU-8 2100

[0254] Before the exposure step, the photomask must be placed over the substrate and aligned with the positive photoresist layer. The exposure energy required depends on the thickness of the photoresist layer, as reported in Table 2.

[0255] Table 2 - Exposure energy (mJ / cm2) v. thickness (pm) of the photoresist SU-8 2100

[0256] In this case, the required energy was 350 mJ / cm2. After this exposure step, a Post Exposure Bake (PEB) heat treatment ensures complete crosslinking of the polymer.

[0257] The PEB in this case was 15 minutes long on a hot plate at 95°C and was optimized according to the thickness of the photoresist layer as reported in Table 3.

[0258] Table 3 - Post Exposure Bake times (min) in relation to the thickness (pm) of the photoresist SU-8 2100

[0259] The development was performed using propylene glycol monomethyl ether acetate (Sigma-Aldrich) for approximately 17 minutes as reported in Table 4. After development, the substrate was rinsed with isopropyl alcohol (Sigma-Aldrich) and then dried with compressed air. A Hard Bake process was then performed on a hot plate at 65°C for 10 minutes and then setting a ramp of 120°C / hour for 2 hours until the final temperature of 160°C.

[0260] Table 4 - Development times (min) in relation to the thickness (pm) of the photoresist SU-8 2100

[0261] 1.3. Soft Lithography for control layer mould

[0262] The control layer mould requires only one photomask. A silicon substrate (2-inch diameter) was washed and dried following the same procedure described in section 1.2. above. The substrate was then inserted into a spin-coater (WS-650-23NPP, Laurell) and secured in the chuck by the vacuum generated by a pump. At this point, a negative photoresist layer SU-8 2050 (Microchem) was distributed and spin-coated over the substrate to obtain a thickness of 35 pm. The spin-coating protocol was: - first step: 500 revolutions per minute for 10 seconds with an acceleration of 100 revolutions per minute / s ;

[0263] - second step: 4000 revolutions per minute for 45 seconds with an acceleration of 300 revolutions per minute / s .

[0264] The duration and temperature of the Soft Bake process step depend on the height of the photoresist layer, as reported in Table 5.

[0265] Table 5 - Soft Bake times (min) in relation to the thickness (pm) of the photoresist SU-8 2050

[0266] In this case the substrate was placed on a hot plate at 95 °C for 6 minutes, before the exposure step. The exposure energy required depends on the thickness of the material as reported in Table 6, and for a thickness of 35 pm corresponds to 160 mJ / cm2.

[0267] Table 6 - Exposure energy (mJ / cm2) v. thickness (pm) of the photoresist SU-8 2050

[0268] The conditions for the Post Exposure Bake step and development depend on the thickness of the photoresist layer as reported in Table 7.

[0269] Table 7 - Post Exposure Bake times (min) in relation to the thickness (pm) of the photoresist SU-8 2050

[0270] The PEB step was performed by placing the substrate on a hot plate at 95°C for 6 minutes. The development with propylene glycol monomethyl ether acetate (Sigma- Aldrich) was performed with the same procedure described in section 1.2. and has a duration of 5 minutes. The final step is the Hard Bake process, with the substrate on a hot plate for 10 minutes at 65°C and a ramp of 120°C / hour for 2 hours in order to reach the maximum temperature of 160°C.

[0271] Table 8 - Development times (min) in relation to the thickness (pm) of the photoresist SU-8 2050 EXAMPLE 2

[0272] Replica moulding

[0273] This section will describe the protocol for producing multilayer microfluidic platforms in PDMS .

[0274] In a first step called "silanization", the substrates of the moulds for the production of the flow and control layer (which correspond respectively to the moulds of layers A and B according to the invention) were treated with trimethylchlorosilane steam (Sigma-Aldrich) for 60 minutes to facilitate the subsequent PDMS demoulding after curing and improve the durability of the mould.. PDMS was prepared by mixing a curing agent (cross-linker) and a base (siloxane) (Sylgard®184, Dow Corning, Michigan, USA) in a 1:10 weight ratio. The solution was then stirred for at least 2 minutes and placed under vacuum in a desiccator to remove air bubbles generated during mixing. The replica moulding process of the PDMS flow layer and control layer follow slightly different protocols. For the flow layer, the PDMS was slowly poured in the centre of the substrate bearing the flow layer mould, made in sections 1.1. and 1.2. above. PDMS was cured in a convection oven at 80°C for 1 hour. Once cooled, the contour of cured PDMS was gently cut with a scalpel and then, starting at the edge of the substrate, was slowly peeled from the mould. The inlets and outlets of the microfluidic platform were made were punched with a dispense tip (Nordson EFD, Ohio, USA) with an external diameter of 0.91 mm.

[0275] The control layer is obtained spin-coating a layer of PDMS over the substrate made in section 1.3. above, to obtain a thickness of approximately 80 pm. The spincoating protocol includes a single 75-second step with an acceleration of 300 revolutions per minute / s and a speed of 1300 revolutions per minute. To increase the pressure resistance during the automation procedure, as well as the adhesion between PDMS and slide, the slide (which corresponds to one of the layers (layer C) of the final platform) is also covered with a PDMS layer 10 pm thick. The spin-coating protocol was the same for the control layer except for the speed, which in this case was 2300 revolutions per minute.

[0276] The slide and the control layer were cured at 65°C for 2 hours. After the heat treatment, the flow layer was attached via plasma activation over the control layer, without peeling the control layer from the mould.

[0277] At this point, the control layer was cut and peeled from its mould and holes were made with the same procedure described above (punched with a dispense tip) for the flow layer. The semi-assembled platform is then attached to the slide, so that the control layer was resting on the slide on the PDMS-bearing surface, with an additional plasma treatment. Finally, the microfluidic platform was placed in the oven at 80 C for at least 10 minutes to facilitate the complete adhesion of the control layer on the slide.

[0278] The microfluidic platform thus produced was made to adhere to a 75x50 cm slide and had the geometry shown in Figure 1. It had the following characteristics described in Table 9.

[0279] Table 9 - Main characteristics of the multilayer platform geometry

[0280] EXAMPLE 3

[0281] 3.1. Installation and verification of the automation and control system

[0282] An electronic unit for generating the signal was connected to an external computer to generate a digital input . Each electrovalve, in particular a solenoid valve, was connected to a tube that is connected to the inlet of the control layer (layer B) , i .e. to the holes of the microvalves, of the microfluidic platforms obtained according to Examples 1 and 2; each tube ends with a needle to be easily inserted into the holes. Each microvalve is connected to a specific electrovalve, which receives a signal independent of the electronic unit . In this way, the opening and closing of the microvalves can be programmed and activated selectively, without interference with the others, ensuring independent and modular flow control. The electrovalves control the flow of compressed air that regulates the open / closed configuration of the microvalves . A pressure regulator was installed: the pressure of the electrovalves can be easily controlled through the pressure gauge and changed through a pressure regulator (CDK Corporation) .

[0283] A programmable syringe pump was connected to the inlets of the main culture chamber and the four secondary culture chambers (one inlet for each chamber) , so as to control the flow of fluid into and out of the chambers. As for the flow of fluid in the microfluidic channels, which connect the main chamber and the secondary chambers, it is controlled by the electrovalve, which activates the microvalves between two configurations, open and closed ones. All the microvalves were set to be driven by electrovalves which are in turn controlled by an electronic unit for the generation of the digital signal (Arduino Uno Rev3 SMD) connected to the USB port of a computer. Each electrovalve can change the microvalves from atmospheric pressure (open position) to the pressure that allows the microvalves to be closed through compressed air. In this way each microvalve can be activated independently of the others, allowing a selective and programmable control of the flow in the different microfluidic channels.

[0284] Once the automation and control system has been implemented, it is necessary to verify the correct operation of the system using a microscope, including the evaluation of the minimum closing pressure for all the microvalves. To do this, the control line pressure was slowly increased from 0 psi, with steps no greater than 1 psi / min, until all the microvalves on the platform opened and closed properly. The syringe pump was used to perfuse the fluids within the microfluidic platform. It is important to set a correct flow rate so as not to damage the microvalves. 5 ml syringes were filled with the desired liquid (dye tracers for the validation of the microfluidic system in dynamic fluid tests, cell suspension in the case of cell seeding, or liquid culture medium in the case of cell culture) , and the tube exiting the syringe was inserted into the inlet of the culture chambers. Finally, the waste tube was inserted into the outlet of the flow layer to collect the waste liquid into a 1.5 ml Eppendorf tube. All the microvalves were then opened using the front panel of the Matlab® code, and all the culture chambers of the microfluidic platform began to be filled with the liquid. If the fluid does not enter certain areas of the platform, air could be trapped inside the PDMS . For this reason, it is necessary to close the microvalves of the platform that control the outlets leaving the microvalves open for entry and wait until the air inside the PDMS permeates therethrough. This procedure is called "debubbling".

[0285] 3.2. Mechanical characterization of the microvalves

[0286] The purpose of this test is to verify the mechanical resistance of the microfluidic platform and its microvalves over the long term. The platform was automated and all platform inlets were connected to the syringe pump by setting an input flow rate of 1 pl / min to ensure a reasonably high flow rate while avoiding platform lamination. The test was performed using different pressure values and was repeated several times for each condition. The tested pressure values ranged from 6 to 25 psi. Each test was performed by setting an automatic program that opens the microvalves for 2 minutes every hour for 1 week.

[0287] Table 10 summarizes the conditions and results of the experiments performed.

[0288] Table 10 - Parameters and results of the microfluidic platform leak test indicates a negative result; "+" indicates a positive result)

[0289] The control pressure was varied from 5 to 25 psi, with 5 psi increments to test different control pressures. The symbol "+" identifies the tests with a positive result, in which the platform has worked correctly throughout the duration of the experiment without lamination or malfunction of the microvalves. The symbol identifies the failed tests. In Experiment 1 the control pressure was not sufficient to completely close the microvalve, while in Experiments 4 and 5 the control pressure was too high: the control layer (layer B) detached itself from the flow layer (layer A) causing the lamination of the platform. The optimal range for the control pressure was therefore between 10 and 15 psi .

[0290] This set of experiments also made it possible to verify the correct operation of the microvalves. Figure 5 shows a representative validation of the flow dynamics with a tracer coloured blue during the closing-opening-closing change of the microvalves. When the microvalve was closed the liquid was confined only in the secondary chamber, when it was changed to the open position the liquid immediately entered the main chamber, and when the microvalve was closed again the flow of the liquid tracer stopped immediately. See Figure 5.

[0291] EXAMPLE 4

[0292] Biological validation

[0293] To verify whether the multilayer platform obtained according to Examples 1 and 2 is suitable for cell culture applications, a biological validation was performed using different cell lines. In particular, the Neuroblastoma cell line SK-N-AS was used to recreate the tumour site, while hMSCs cells (human mesenchinal stem cells) and HUVEC cells (human umbilical vein endothelial cells) were used as representative of the target metastatic sites of the Neuroblastoma.

[0294] 4.1. Cell seeding and culture within the automated microfluidic platforms

[0295] Firstly, the workstation was prepared by configuring the PC, incubator, syringe pump, and microscope.

[0296] To seed and grow the cells, the experimental set-up included :

[0297] A) An automation and control system interfaced with a computer using an appropriate Matlab® code and solenoid valves connected through tubes to the inlets (holes) of the microfluidic platform;

[0298] B) a syringe pump to seed the cell solution in the platform and perform the change of liquid culture medium. Each syringe was connected to the correct inlets in the flow layer (layer A) of the platform;

[0299] C) a portable incubator to maintain platforms at standard cell culture conditions for the entire duration of the set-up procedures and experiments.

[0300] The test was repeated on multiple platforms. In this section, for simplicity of exposure, only one platform will be referred to.

[0301] The microfluidic platform was placed inside a Petri dish and sterilized by UV. The microfluidic platform was then connected to the automation and control system and the minimum pressure to completely close all the microvalves inside the platform was verified. The microvalves were then all opened. The "debubbling" was then performed to remove all air bubbles with a 5 ml syringe filled with a saline solution and connected through a tube to an inlet of the microfluidic platform. The pump flow rate was set to 4 pl / min and the saline (aqueous) solution was perfused through the platform (keeping the microvalves open) until the platform was completely filled with liquid. At this point, a coating treatment of the surface of the culture chambers was performed to improve cell adhesion using fibronectin (Thermo Fisher Scientific, Waltham, MA, USA) , an extracellular matrix glycoprotein, at a concentration of 25 pg / ml. A syringe was filled with 1 ml of fibronectin solution which was injected at a flow rate of 2 pl / min into the chambers and incubated for 1 hour at room temperature (25°C) . All microvalves were then closed to begin the cell seeding step. The tumour SK-N-AS cells were seeded only in the main chamber, while two secondary chambers were seeded with hMSC cells and two secondary chambers with HUVEC cells. The different cell lines were seeded in the platform at an optimized final density of:

[0302] • 200 cells / mm2for SK-N-AS cells;

[0303] • 100 cells / mm2for hMSC cells;

[0304] • 150 cells / mm2for HUVEC cells.

[0305] Different syringes were filled with the cellular solutions and connected to the appropriate inlets (SK- N-AS cells in the main chamber, hMSC cells and HUVEC cells in the secondary chambers) , the microvalves were closed, and the pump flow rate was set to 2 pl / min by perfusing the cellular solution for about 10 seconds. This operation is carried out for all chambers. The procedure was completed by placing the platform in a 37°C incubator while keeping the microvalves closed. After 24 hours from seeding, each inlet for liquid culture medium replacement was connected to the syringe pump. During this step, the microvalves were kept closed, and switched to the open position when the replacement of the liquid culture medium was complete and communication between the different culture chambers was allowed .

[0306] 4.2. Essay on viability and mortality

[0307] To establish the viability of the cells, a Live&Dead viability assay was carried out 48 hours after seeding. To perform this test, different microfluidic platforms were seeded using three different cell lines, namely HUVEC, hMSCs and SK-N-AS, the cells were cultured for 48 hours in the incubator at 37°C, disconnected from the automation system, and finally in each chamber a solution of Hoechst and Calcein-AM was introduced as fluorescent markers. In particular, two platforms were used for the co-culture of SK-N-AS and hMSCs and one platform for SK-N-AS and HUVEC. In the aforesaid platforms, the SK-N-AS line was introduced in the main chamber, while hMSCs and HUVEC were introduced in the four secondary chambers, keeping the microvalves closed.

[0308] The labelling solution was prepared as follows .

[0309] A solution of Hoechst 33342 (Sigma Aldrich) in PBS in concentration 1:1000, and a solution of Calcein-AM (Sigma-Aldrich) in PBS in concentration 1:500 which were added for cellular staining were used.

[0310] After washing the platforms with PBS to remove the liquid culture medium containing foetal bovine serum (FBS) , the previously prepared solution was manually pipetted into the platforms, which were then placed in the incubator at 37 °C for 15 minutes. Then, the platforms were analysed with a fluorescence microscope and images were taken for subsequent counting and calculation of cell viability by image analysis with ImageJ.

[0311] 4.3. Staining of the co-culture with Phalloidin and DAPI Different cell lines were seeded in each platform: SK- N-AS / HUVEC cells and SK-N-AS / hMSC cells, according to the scheme of Figure 9. The cells were cultured in the incubator at 37°C for 72 hours keeping the microvalves open, except at the time of changing the liquid culture medium in which the microvalves were closed, and then disconnected from the automation system and analysed with the microvalves open. The cells were fixed in 4% formaldehyde (Sigma-Aldrich, F8775) for 25 minutes at room temperature (25°C) . After formaldehyde removal, the cells were washed three times with IX PBS. A 0.1% solution of Triton X-100 in PBS was prepared and injected into the microfluidic platform (taking care not to introduce bubbles) . The platforms were then allowed for 15 minutes at room temperature (25°C) to permeabilize the cell membranes and then washed numerous times using IX PBS. A solution of tet ramethylrhodamine-con ugated Phalloidin (TRITC) (1:400, Sigma-Aldrich) was pipetted into each platform which were incubated for 50 minutes at room temperature: the platforms were then washed with PBS and incubated with a 1:1000 DAPI solution for 15 minutes. Phalloidin is a cyclic peptide with high affinity to tetramethylrhodamine-con jugated F-actin (TRITC) filaments, which emits red-orange light when excited by the green light of a fluorescence microscope.

[0312] Phalloidin binds to F-actin with high selectivity while TRITC provides the bright red-orange fluorescence and photostability. DAPI is a blue fluorescent marker that selectively fluoresces for DNA with high cellular permeability and allows efficient staining of the nuclei. At this point the microfluidic platform was observed and analysed using a fluorescence microscope.

[0313] 4.4. Staining of Immunofluorescence co-culture ( IF ) To verify the effects of the co-culture experiments on SK-N-AS and hMSC cells, cells were analysed by immunofluorescence staining performed within a microfluidic platform.

[0314] The SK-N-AS / hMSC cell lines were seeded in a platform according to the scheme of Figure 9a. The test was repeated several times. In the following, only one platform will be referred to.

[0315] After 72 hours of cell culture in the incubator at 37°C with the microvalves open, the platform was disconnected from the automation system and the cells were fixed in 4% formaldehyde (Sigma-Aldrich, F8775) for 25 minutes at room temperature (25°C) with the microvalves open. After formaldehyde removal, the cells were washed three times with IX PBS. After that the cells were incubated for 1 hour with the blocking solution composed of PBS- / - with 5% bovine serum albumin (BSA) (Sigma-Aldrich) and 0.1% Triton X-100. After 1 hour, the platform was washed three times with a solution of PBS- / - with 0.1% Triton X-100 (PBST) . It was decided to investigate the phenomenon of epithelial-mesenchymal transition (EMT) in metastasis and tumour invasion. The fact that cells can downregulate epithelial features and acquire mesenchymal traits is a key step during embryonic development; similarly, epithelial tumour metastases can change cell-to-cell and cell-extracellular matrix interactions by altering cellular molecular levels, causing cell transmigration, and ultimately leading to metastasis. For this reason, Vimentin protein expression was assessed both in SK-N- AS and hMSCs cells when co-cultured in the microfluidic platform. Vimentin is a multifunctional protein and its ability to interact with a large number of proteins makes it a potential regulator of various physiological functions. This protein is responsible for the maintenance of cell shape, the integrity of the cytoplasm, the stabilization of cytoskeletal interactions, and involves an immune response. Vimentin expression is mainly associated to metastatic phenotype and poor prognosis of disease outcome. This antibody is only used for hMSC cells at a dilution of 1:100. The primary antibody was prepared in blocking solution (PBS- / - containing 5% BSA and 0.1% Triton X-100) . The primary antibody was applied overnight at 4 C, then the cells were washed with PBST and incubated for 1 hour with the secondary antibody of choice. Alexa Fluor 488 goat Anti- Rabbit is used at a dilution of 1:1000 for Vimentin. After 1 hour, the cells were washed with PBST for 2 times and with PBS- / - once. The cell nuclei were then counterstained with a solution of DAPI ( 4 ' , 6-diamidine- 2-phenylindole) for 10 minutes. The cells were then washed with PBS- / - and all platforms were filled with mounting medium composed of 80% Glycerol and 20% Milli- Q water. Finally, the samples were observed and analysed with a confocal fluorescence microscope. Image analysis was performed using ImageJ software. This method allows to quantify the IF data collected on a confocal microscope by quantifying the relative levels of a molecule of interest by measuring the mean fluorescence intensity (MFI) across a region of interest. The cell samples were observed with a 40x objective using two different dyes: Alexa 488 for the green channel (Vimentin) and Dapi for the blue channel (cell nuclei) . The quantification of the images was done using ImageJ; in particular each image was converted into 16 bits and the fluorescence intensity was measured keeping the threshold constant for all the data collected for each antibody staining.

[0316] 4.5. Results

[0317] To mimic the metastatic behaviour of neuroblastoma (NB) from the primary tumour to the various organs, the corresponding cell lines were used to reconstitute in vitro both the primary Neurobloast oma tumour (SK-N-AS) and its metastatic site microenvironments (hMSCs for bone marrow and HUVECs for vasculature) .

[0318] Each cell line was seeded at different and optimized densities: SK-N-AS cells at 200 cells / mm2, HUVEC cells 150 cells / mm2and hMSC cells 100 cells / mm2, all using the automation system described above.

[0319] 4.5.1. Essay on viability and mortality

[0320] Cell viability was evaluated during simultaneous culture of the different cell lines 48 hours after seeding using detection with Calcein-AM fluorescence labelling live cells (green) and Hoechst fluorescence for all nucleated cells (blue) , as described in section 4.2.. Live cells were quantified by image analysis (ImageJ) and measured as consistently high, demonstrating proper maintenance of the appropriate culture conditions within the platform .

[0321] Figure 6 shows the results of the viability tests for SK-N-AS cells, with image analysis allowing an average viability of 85.83 ± 3.97% to be calculated.

[0322] Figure 7 refers to the results obtained for the hMSC cells 48 hours after seeding, the measured viability is here 97.45 ± 2.37%.

[0323] Similarly, Figure 8 shows the results of the viability tests for the HUVEC cells 48 hours after seeding; image analysis results in a viability of 97.05 ± 1.43%.

[0324] 4.5.2 Staining of the co-culture with Phalloidin and DAP I

[0325] In this experiment, following the protocol described above in section 4.3, the platforms were seeded with different cell types following the scheme reported in Figure 9.

[0326] The cells were cultured for 72 hours in a fully automated manner and then fixed and labelled following the experimental protocol described to observe their morphology and behaviour under co-culture conditions. As already mentioned, the Neuroblastoma SK-N-AS cells were seeded in the main chamber (and represent the main tumour) , while the HUVEC or hMSC cells occupy all the secondary chambers to recreate the tumour microenvironment and the target metastatic site.

[0327] Figure 10, Figure 11 and Figure 12 report representative results after labelling with DAPI and Phalloidin for both experiments (after 72 hours) . From the images it can be observed how the Neuroblastoma cells after 72 hours of co-culture seem to form and grow in clusters. It has been observed that the growth of SK-N-AS cells in clusters occurs both for the Neuroblastoma cells that reach and settle in the secondary chambers (due to the convective flux and / or their migration) , and for the Neuroblastoma cells present in the main chamber whose behaviour is affected by the chemical and biological signals of the other cells co-cultured in the same platform. Many studies confirm that adjacent tumour cells can use defence mechanisms to protect themselves by secreting specific substances. The microenvironment of the tumour cells can be affected by interactions with other cells that are mediated by secreted chemokines, and other proteins (e.g. , proteases) . The action of secreted factors on the microenvironment may also facilitate tumour cell survival and progression. Another protection mechanism adopted by tumour cells is the formation of clusters: "autologous" intercellular junctions can cluster heterogeneous sub-clones in invasion through a basement membrane or endothelium.

[0328] Once a potential metastatic sub-clone undergoes a metastatic change, it can take advantage of the prior conditioning of the microenvironment induced by the other tumour cells. In particular, it can be observed in Figure 11 and Figure 12 that the tumour cells (typically smaller in size and rounder in shape) in the secondary chambers grow as clusters that are completely surrounded by the other cells.

[0329] 4.5.3. Immunofluorescence results of the co-culture

[0330] After staining with specific antibodies and acquisition with the Confocal microscope, the images were analysed by measuring the MFI by ImageJ. The statistical analysis was performed using GraphPad software through a Student t-test. Asterisks indicate a significant difference between the treated and control group, unless otherwise specified (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) . Figure 13 shows representative images at 20X magnification and shows the morphology of the cells inside the platform 72 hours after seeding.

[0331] For Vimentin, the mean fluorescence intensity was calculated for SK-N-AS and hMSC cell co-cultures, and together compared to the cells cultured alone. Representative images of Vimentin protein expression in the SK-N-AS cells are reported in Figure 14: from visual inspection alone, the difference in fluorescence intensity expressed by the Neuroblastoma cells in the two conditions is clearly visible. Representative images of Vimentin expression in hMSC cells for both tests are reported in Figure 15. Also in this case, there is a difference in fluorescence intensity. Figure 16 and Figure 17 show the results of MFI quantification, confirming that when Neuroblastoma cells and hMSCs are cultured together, there is a significant change in Vimentin protein expression. Both cell lines, when in co-culture conditions, show an increase in Vimentin expression compared to the control experiment.

[0332] These results indicate that the microenvironment induces overexpression in the proteins involved in cancer progression and aggressiveness in both the SK-N-AS tumour cells and the healthy ones of the hMSCs target site. Furthermore, the results highlight that the microenvironment for co-culture conditions induces an upregulation of Vimentin, a protein that is directly involved in EMT and proliferation. See Figures 16 and 17.

[0333] Thanks to the cell culture method of the present invention, which uses the platform and the system of the invention, it is possible to observe the growth of the tumour cells, for example in the main culture chamber, and to observe the migration thereof towards the target metastatic present in the corresponding secondary culture chamber (s) .

[0334] The microfluidic system of the present invention thus represents a valid and improved tumour and metastasis model and can be used to observe the progression of a tumour and to test drugs / active ingredients potentially useful for the treatment of cancer.

[0335] The microfluidic system of the invention has the following characteristics: simplicity and reproducibility, reliability and durability, ease of automation and integration with different analysis and image processing techniques. The moulds (for the different layers) of the microfluidic platform are manufactured with soft lithography techniques, and the platforms produced using the replica moulding technique. This allows to obtain an easily reproducible structure in a short time. The protocol for the automation of the multi-layer platform is simple and easily implemented, and the automation software is easily usable by the user and allows several parameters to be easily set. Biological validation showed that the cells adhere correctly and grow within the multilayer platform for up to 7 days, suggesting that the microfluidic system is suitable for biological applications. Advanced on-chip technology has shown success in enabling simultaneous culturing of live and physiologically active cell populations, which constitute the primary tumour and its target metastatic sites. The Examples refer to the behaviour of Neuroblastoma and the interaction with its target metastatic sites within a single multilayer platform but can easily be used for different tumours. The microfluidic system and the microfluidic platform of the invention enable integrated studies of multiple aspects of the metastatic microenvironments with a high level of control and experimental flexibility.

[0336] This result constitutes an improvement over the prior art. In particular, the microfluidic systems of the prior art are not able to vary the experimental conditions in real time, such as for example the flow of the fluid or the communication between the different cell culture areas present on the chip at the same time. The microfluidic platform of the present invention, as well as the system comprising it, allows the study of the cells in both a two-dimensional (where the cells form a monolayer) and a three-dimensional (where the cells, for example in the presence of a gel, occupy all the space available in the culture chambers) environment. The two options are applicable to the same platform, without the need to make specific changes.

[0337] The microfluidic system of the present invention represents a successful compromise between a great complexity of geometry and functions and ease of use by the operator.

Claims

CLAIMS1) Multilayer microfluidic platform (2) suitable for cell culture comprising:- at least one main culture chamber (3) and at least one corresponding secondary culture chamber (4) , said at least one main culture chamber (3) and said at least one corresponding secondary culture chamber (4) being in fluid communication with each other;- at least one microfluidic channel (5) adapted to put in fluid communication said at least one main culture chamber (3) with said at least one corresponding secondary culture chamber (4) ; and- at least one microvalve (10) associated to at least one said at least one microfluidic channel (5) , said at least one microvalve being actionable to modify, at the site of said at least one microvalve, the cross-section of said at least one microfluidic channel between an open configuration in which the area of the cross-section of said at least one microfluidic channel (5) is maximum and a closed configuration in which the area of the cross-section of said at least one microfluidic channel (5) is zero.2) Automated microfluidic system (1) suitable for cell culture comprising: a) a multilayer microfluidic platform (2) suitable for cell culture comprising:- at least one main culture chamber (3) and at least one corresponding secondary culture chamber (4) , said at least one main culture chamber (3) and said at least one corresponding secondary culture chamber (4) being in fluid communication with each other;- at least one microfluidic channel (5) adapted to put in fluid communication said at least one main culture chamber (3) with said at least one corresponding secondary culture chamber (4) ; and- at least one microvalve (10) associated to at least one said at least one microfluidic channel (5) , said at least one microvalve being actionable to modify, at the site of said at least one microvalve (10) , the crosssection of said at least one microfluidic channel between an open configuration in which the area of the crosssection of said at least one microfluidic channel (5) is maximum and a closed configuration in which the area of the cross-section of said at least one microfluidic channel (5) is zero; b) an automation and control system (11) adapted to control the activation of said at least one microvalve (10) between said open configuration and said closed configuration; and c) optionally at least one programmable pump (13) in fluid communication with said at least one main culture chamber (3) and / or said at least one corresponding secondary cell chamber (4) .3) Multilayer microfluidic platform (2) according to claim 1 or automated microfluidic system (1) according to claim 2, wherein said at least one microvalve (10) associated to said at least one microfluidic channel (5) is a push-up microvalve.4) Multilayer microfluidic platform (2) or automated microfluidic system (1) according to any one of claims 1 to 3, wherein said at least one main culture chamber(3) and said at least one corresponding secondary culture chamber (4) each comprise at least one inlet (6, 8) and at least one outlet (7, 9) , wherein preferably said at least one inlet (6, 8) is adapted for the introduction of a fluid in said at least one main culture chamber (3) or in said at least one corresponding secondary culture chamber (4) , and said at least one outlet (7, 9) is adapted to remove a fluid from said at least one main culture chamber (3) or from said at least one corresponding secondary culture chamber (4) .5) Automated microfluidic system (1) according to any one of claims 2 to 4, wherein said automation and control system (11) comprises:- at least one electrovalve in fluidic communication with said at least one microvalve (10) , adapted to activate said at least one microvalve ;- a pressure regulator in fluidic communication with said at least one electrovalve; and- a computing and control unit in data communication with said at least one electrovalve and said pressure regulator.6) Multilayer microfluidic platform (2) or automated microfluidic system (1) according to any one of claims 1 to 5, wherein said at least one microfluidic channel (5) has a maximum width from 120 pm to 300 pm, preferably from 150 to 250 pm, more preferably 200 pm.7) Multilayer microfluidic platform (2) or automated microfluidic system (1) according to any one of claims1 to 6, wherein at least one of said main culture chambers (3) is in fluid communication with at least two, preferably from two to ten, more preferably from two to six, even more preferably four, of said corresponding secondary culture chambers (4) .8) Multilayer microfluidic platform (2) or automated microfluidic system (1) according to any one of claims 1 to 7, wherein at least one of said main culture chambers (3) is surrounded by four corresponding secondary culture chambers (4) , which are at the vertices of a square whose diagonals intersect at the centre of the main culture chamber (3) .9) Multilayer microfluidic platform (2) or automated microfluidic system (1) according to any one of claims 1 to 8, wherein said microfluidic platform (2) comprises a first level comprising said at least one main culture chamber (3) , said at least one corresponding secondary culture chamber (4) , and said at least one microfluidic chamber (5) ; and a second level comprising said at least one microvalve (10) .10) Cell culture method, comprising the steps of: a) providing said automated microfluidic system (1) comprising said multilayer microfluidic platform (2) , according to any one of the previous claims; b) introducing at least one fluid in said at least one main culture chamber (3) and / or in said at least one corresponding secondary culture chamber (4) ; and c) activating said automation and control system (11) ,preferably by way of a suitable set program.11) Method according to claim 10, wherein in step b) said fluid is chosen from cell suspension, liquid culture medium, and mixtures and combinations thereof, more preferably cell suspension.12) Method according to claim 10 or 11, wherein step b) comprises the introduction in said at least one main culture chamber (3) of a cell line of tumour cells, preferably chosen from cell line of neuroblastoma cells, cell line of glioblastoma cells, cell line of rectal colon tumour cells, cell line of ovarian tumour cells, preferably cell line of neuroblastoma cells, more preferably SK-N-AS cell line of neuroblastoma cells.13) Method according to any one of claims 10 to 12, wherein step b) comprises the introduction in said at least one corresponding secondary culture chamber (4) of a cell line representing the target metastatic site of a tumour cell, preferably chosen from cell line of human mesenchinal stem cells (hMSC) and cell line of human umbilical vein endothelial cells (HUVEC) .14) Method for verifying the metastatic migration of a cell line of tumour cells, comprising the steps of: a) providing said automated microfluidic system (1) comprising said multilayer microfluidic platform (2) , according to any one of claims 1 to 9; b) introducing said at least one fluid in said at least one main culture chamber (3) and / or in said at least one corresponding secondary culturechamber (4) ; and c) activating said automation and control system (11) , preferably by way of a suitable set program; wherein in step b) a cell line of tumour cells is introduced in said at least one main culture chamber (3) and a cell line representing the target metastatic site of the cell line of tumour cells is introduced in said at least one corresponding secondary culture chamber ( 4 ) ; and d) detecting the possible presence of metastasis of the cell line of tumour cells in said at least one corresponding secondary culture chamber (4) .15) Method according to claim 14, wherein step d) is carried out by way of a fluorescent staining technique, preferably chosen from a viability assay, PCR and immunofluorescence .16) A method for testing the influence of a test substance apt to induce a cellular response, preferably a drug or an active ingredient, on a cell culture, comprising carrying out a cell culture method according to any one of claims 10 to 13, wherein step c) comprises putting in contact said cell culture with said test substance, incubating in controlled conditions, and evaluating the effect of the substance, preferably by way of a fluorescent staining technique.17) Use of the multilayer microfluidic platform (2) or of the automated microfluidic system (1) according to any of claims 1 to 9 for cell culture, for the simulationof the metastatic process; for the pharmaceutical screening of active ingredients or drugs destined to the treatment of a tumour, preferably solid or liquid tumours; for the evaluation of the effects of molecules and / or nanoparticles; and / or for drug discovery.

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