IT202100025460A1 - LAB-ON-CHIP DEVICE TO STUDY CELLULAR MIGRATION IN THREE-DIMENSIONAL SYSTEMS AND RELATED METHOD OF USE - Google Patents

Description

Description of the industrial invention entitled: LAB-ON-CHIP DEVICE TO STUDY CELLULAR MIGRATION IN THREE-DIMENSIONAL SYSTEMS AND RELATED METHOD OF USE;

DESCRIPTION

Field of application

In its most beautiful aspect In general, the present invention concerns microfluidic chip technology, or Lab-on-Chip, for the study of cell biology processes in three-dimensional (3D) systems, such as cell migration, with application in the biomedical and diagnostic fields. In the following, this device will be also referred to as 3D cell-migration chip (3D-CM chip).

Known technique

Cell migration plays a crucial role in several biological processes, including embryogenesis, wound healing in a biological tissue, or cicatrization, cell differentiation, tissue development, angiogenesis, and metastasis, just to mention a few . Cells are stimulated to migrate in response to soluble factors, chemoattractants or chemorepulsors, proinflammatory molecules released in many physiological and pathological states; the activity? of research on the process of cell migration? fundamental for shedding light on the molecular mechanisms underlying many conditions and identifying the key elements on which to direct therapeutic and/or diagnostic action.

In general, cell migration is stimulated and directed by the interaction of cells with the extracellular matrix (ECM), neighboring cells or chemoattractants. The process of cell migration requires the integration and coordination of signaling events and changes in cellular architecture. ? a cyclic process in which a cell extends its protrusions at its front, the structure is stabilized by the formation of adhesive complexes, and then retracts at the end posterior, allowing the cell to advance on its substrate. The adhesiveness? cell-substrate plays a fundamental role in determining the speed? of migration. Cell migration in higher organisms? mediated by these adhesions, which transmit forces and signals necessary for motility? mobile phone. If the cells migrate on a certain substrate, their respective speed? depends on several variables related to integrity? and to the interactions that include the density? of the ligand surface, integrin levels and affinity? of bond. Therefore, techniques that allow the quantification and visualization of the elements involved in this complex pattern are also fundamental to clarify the key elements of cell migration and the many cellular processes in which ? involved.

Conventional cell migration assays generally require large amounts of of cells and large volumes of reagents, and this makes them unsuitable for high-throughput screening. To overcome this problem, on-chip cell migration assays using microfluidic channels have been developed.

Microfluidics? the science of high-precision control and manipulation of fluids that are geometrically forced into networks of small channels, normally between 100 ?m thick and 1 ?m in diameter.

The structure consists of the parts that guide the movement of fluids through the channel system with a diameter ranging in size from 100 ?m to 1 ?m ? normally called a "chip".

The microchannels present in the chip allow the manipulation of fluids in large quantities. down to a few picoliters and the manipulation of biochemical reactions at very small volumes. Of course, to enable all these operations, the devices are not only a collection of microchannels, but are also equipped with integrated pumps, electrodes, valves, flow control systems, electric field generators and electronics that regulate the movement of fluids, sometimes they also create gradients of substances. This way in a miniaturized device ? It is possible to create cellular environmental conditions by integrating into a single chip one or more analyses, which are usually performed in the laboratory and become complete "lab-on-a-chip" study and analysis systems.

The rationale for lab-on-a-chip? integrate on a single chip thousands of biochemical operations that could be performed by dividing a single drop of blood taken from the patient to obtain a precise diagnosis of potential diseases or by subjecting a sample of cells to various analyzes with various substances or in various conditions even at the same time showing also the related interactions.

Thanks to the microfluidic approach? Was it possible to expose colonies of human pluripotent stem cells (hPSCs) to spatiotemporally controlled gradients of morphogens generated by artificial signaling centers that can extrinsically control capacity? endogenous stem cell self-organization. An application example of such an approach demonstrated that in response to a localized source of bone morphogenetic protein 4 (BMP4), hPSC colonies reproducibly break their intrinsic radial symmetry to produce distinct, axially arranged differentiation domains. Lab-on-chip analysis further showed that the antagonistic action of the NOGGIN protein improves this spatial control of cell fate patterning and morphogen concentration and cell density. cell influence the BMP response and germ layer patterning [Manfrin A., et al. Engineered signaling centers for the spatially controlled patterning of human pluripotent stem cells. Nature Methods 2019, 16: 640?648].

Nie F. Q. et al. describes an assay conducted in an on-chip cell migration device equipped with a microfluidic channel, consisting of a central chamber with three inlets. After the cells have been cultured in the central chamber, a solution of trypsin ? made to flow through two inlets, the passage of the solution causes the removal of cells from a portion of the channel creating a discontinuity? in the layer of cells that mimics the edge of a wound, it is thus activated? the healing response which involves the migration of cells towards the empty area. However, the method concerns a two-dimensional migration analysis and does not allow chemical analysis of the substrate surface [Nie, F. Q., et al. On-chip cell migration assay using microfluidic channels. Biomaterials. 2007; 28:4017-22].

International patent application WO 2009/126524 describes a system for high-throughput analysis comprising one or more? microfluidic devices, each comprising an optically transparent material; a substrate coupled to the optically transparent material; and a scaffold having dimensions of at least 1 ?m in three-dimensional space ? interposed between two non-intersecting fluid flow paths; the scaffold comprises solid or semi-solid biocompatible polymers that allow the passage of one or more entity? biological. The method using such a device to study cell migration includes: (a) providing a device of the type described, (b) introducing a quantity? defined of cells in the first path of fluid flow; (c) introduce an entity? biological, for example a chemoattractant, in the second fluid flow path; and (d) measure the directed migration of the cell into the scaffold;

International patent application WO 2012/050981 describes microfluidic devices that represent an improvement over existing devices as they allow for more simple determination of the image with quantitative and not only qualitative data of the result, they can be used as a 3D biological assay. The device ? a small block made of optically transparent material, such as glass, plastic or a polymeric material, such as polydimethylsiloxane, polymethacrylate, polystyrene, cyclic olefin copolymer, comprising a series of small fluid channels in which one or more channels can be filled with a biologically relevant gel, held in place with pins. The gel in which endothelial cells are seeded and cultured is thick enough to allow cells to grow in the three-dimensional space of the gel. Through the other fluid channels present in the system, ? Is it possible to add drugs or biological material that can interfere with growth and motility? 3D cell phone. By observing the growth and migration of cells on the surface of the 3D gel structure, the device allows you to measure rates of angiogenesis, as well as the intervascularization and extravascularization of cancer cells.

Although the use of such devices with application to the study of cell migration is widespread, the need is still strongly felt. of improved devices and related methods of use that allow for greater in-depth and precise analysis of cellular interactions and reactions to the use of possible therapeutic agents.

Recent technological advances in materials design and polymer synthesis have made it possible to develop new materials and matrices with a?heterogeneity? space within systems that can incorporate cultured cells, opening up the possibility of to create organotypic screening platforms with increasingly more control methods? precise details of cell-cell and cell-biomaterial interactions [Friedrich, J., et al. Spheroid-based drug screen: considerations and practical approach. Nat Protoc.

2009; 4:309-24.; Kloxin, A. M., et al. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science. 2009; 324:59-63; Lutolf, M. P. et al. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol. 2005; 23:47-55. Seliktar, D. Designing cell-compatible hydrogels for biomedical applications. Science. 2012; 336:1124-8]. Thanks to gelation processes compatible with cell survival, such as photopolymerization,? It is now possible to create scaffolds that mimic the extracellular matrix and the three-dimensional physiological environment, providing optimal conditions for the creation of 3D growth systems for stem and tumor cells.

Ivanir, E., et al. [A Gel?Based Model of Selective Cell Motility: Implications for Cell Sorting, Diagnostics, and Screening 2020, Advanced Functional Materials, 30, 18] describes an ?on plate? (on plate) used to study cell migration in three-dimensional hydrogel-based systems, capable of mimicking the extracellular matrix (ECM). It is a three-dimensional "gel in gel" system, based on PEG-fibrinogen (PFHy), containing the cells (sphere with cells) embedded in an external gel with different composition and properties. (sorting gel). Microgels are produced by immobilizing cells in a PEGylated fibrinogen precursor solution with cross-link formation via a radical photopolymerization reaction [L. Almany, D. Seliktar, Biomaterials 2005, 26: 2467-77]. The gel spheres are immersed in a precursor solution of the external gel and the polymerization of the latter is carried out inside the multiwell. The chemical composition and biological nature of the two gels, so? like their relative positioning, they provide a framework for creating intricate spatio-temporal scenarios with living cells in situ. The cells found in the internal gel migrate in response to external stimuli and tend to invade the external gel at different speeds. and with different motility mechanisms. Through the modulation of the properties? of the external hydrogel and the consequences on motility? cells provide quantitative response information to potential therapeutic treatments [Ivanir, E., et al. A Gel?Based Model of Selective Cell Motility: Implications for Cell Sorting, Diagnostics, and Screening 2020, Advanced Functional Materials, 30, 18].

The "gel in gel" system, while demonstrating its validity of the migration assay, has some important limitations that do not make it easy to use by inexperienced personnel. In particular, the system involves the creation of gel spheres including cells using insulin syringes, with the production of spheres with dimensions of 20-50 ?L, variable within the same preparation. Furthermore, the passage of cells through the syringe needle can lead to mechanical damage resulting in cell death, therefore? density required? elevated cell in the preparation of the internal sphere. The spheres are made on plates whose surface had been functionalized with colloidal silica to make it superhydrophobic and subsequently transferred into multiwells or dishes. This step involves the difficulty? to transfer intact spheres without damage or, in some cases, fragmentation of the spheres. After incubation for 24 hours in culture medium, the spheres are transferred to a multiwell, where the second gel is produced by photopolymerization. Since? between the two phases? It is necessary to wash the spheres with a swab to eliminate the soil, which hinders the photopolymerization process, very often it is possible? having the spheres break resulting in loss of the sample.

Summary of the invention

The present invention addresses the foregoing and other prior art needs by providing a device to facilitate the conduct of an in vitro cellular assay that can be used to study the effects of the administration of biological or therapeutic agents interfering with the process of cell migration. In particular, the device according to the invention? a microfluidic device that allows cell growth and development in 3D. This device represents a significant improvement over currently available devices.

A further aspect of the present invention concerns the method of use of the device.

A better understanding of the invention will be had? with the following detailed description and with reference to the attached figures which show, purely by way of example and not already? limiting, a preferred embodiment.

Brief description of the figures

Figure 1: shows an exemplary 3D diagram of a preferred embodiment of the invention;

Figure 2: ? a top plan view of the invention, which also shows some measurements purely by way of example;

Figure 3: ? a further 3D schematic view of the invention in fig. 1, relating to the main components;

Figure 4: shows an exploded view, a 3D view, a plan view and a side view of a construction example of the invention, created using several layers superimposed on each other, but it could also be made by 3D printing, or thermoplastic molding, or other construction technique;

Figures 5 and 6: are photographs of an experimental prototype, with and without a culture liquid respectively;

Figures 7 and 8: are photographs that show enlarged details of the experimental prototype in fig. 5-6;

Figure 9: image obtained by inverted bright field microscope with 4X magnification of the microsphere of MDA-MB 231 cells in PEG-fibrinogen hydrogel detected by 3D-cell migration chip. A: T=0, B: T=3 days of incubation, C: T=7 days of incubation.

Figure 10: image obtained by inverted bright field microscope with 4X magnification of the microsphere of Lin-Sca-1<+ >hcMSC cells in PEG-fibrinogen hydrogel detected by 3D-cell migration chip. A: T=0, B: T=3 days of incubation, C: T=5 days of incubation.

Detailed description of the invention Main purpose of the present invention? provide a microfluidic device that can? be easily used in a 3D bioassay, to study the process of cell migration in vitro on a miniaturized scale, and for the screening of biological agents and/or drugs that directly or indirectly intervene in cell migration, wound healing, tumor growth and metastasis, and in other processes in which? involved cell migration.

The device represents an improvement over currently available devices and essentially consists of a block of polymeric material with regions, channels and/or chambers for the flow and/or containment of fluids and/or gels, typically on a miniaturized scale (e.g. microliter , nanoliter, picoliter). In order to facilitate the visualization of the contents, the microfluidic device ? typically consisting of a substrate transparent to light, or an optically transparent material, such as polymer, plastic and glass. Examples of biocompatible, degradation-resistant, and light-transparent polymers and suitable laser beams are polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA), polystyrene (PS), polyethylene glycol diacrylate (PEGDA).

The device object of the invention can? advantageously be produced via extrusion 3D printing, photolithography or casting.

In the preferred embodiment of the invention described, said substrate is substantially constituted by a block of optically transparent material which includes one or more regions, each shaped like a well for containing a fluid, gel, hydrogel or similar. Each well P can? have a variable shape depending on the use you want to make of it, for example it can? have a cylindrical shape with a height h=4-6 mm; a diameter d=4-6 mm; and a volume V=50-169 ?L. In the preferred embodiment described, the height is of 5 mm and the diameter? of 5 mm with an internal volume of 98.2 ?L.

In the illustrated example the device includes three wells, each of which is suitable for containing a fluid, gel, hydrogel or similar, which constitute the gel-in-gel system, consisting of a composite matrix of natural biomaterials, which creates a structure that has the capacity to modulate the development, organization and final function of a multicellular system, a potential scaffold for cell culture. The gel-in-gel system can? be made up of different types of both thermo- and photo-polymerizable polymers, for example agarose, gelatin, fibrin, collagen, chitosan, and double gelatin and PEG-fibrinogen systems. In a particularly preferred embodiment of the invention, the well contains a gel-in-gel system, based on PEG-fibrinogen (PFHy).

Specifically, the gel-in-gel system contained in the device according to the invention is essentially consists of a 1-10 ?L gel microsphere containing cells at a specific density? (>2000-cells/?L) which is placed at the base of the well using a micropipette. The gel in which the cells are seeded/trapped constitutes the internal portion of the gel-ingel system, or internal gel, which is generally made up of a hydrogel and therefore has a hydrophilic character, however depending on the nature of the hydrogel used, depending on the experimental needs, its degree of hydrophilicity/hydrophobia can? to vary. AND? Furthermore, it is clear that the shape of the gel and the distribution of the cells within it, at the beginning of the experiment, are of fundamental importance for determining the correct parameters of cell migration and the interactions that we intend to study. The deposition of the gel will go? to constitute the internal portion of the gel-in-gel system? influenced in turn by the nature of the material of which the device is made? constituted: in contact with a hydrophilic surface a portion, for example a drop, of gel or medium, consisting of an aqueous medium, tends to diffuse, vice versa, in contact with a hydrophobic surface, the gel or any hydrophilic medium tends to take on a substantially spherical shape.

Depending on the system you intend to analyze, you can It may be necessary to have a substantially spherical shape of the internal gel.

According to the present invention, in order to obtain the deposition of a gel with a substantially spherical shape, a removable superhydrophobic surface 3 is provided on the bottom of the well, on which the gel is deposited. According to this description, a surface ? hydrophobic when a drop of water forms a contact angle greater than 90 with the surface, if this angle is above 150? the surface ? called superhydrophobic, favoring the formation of isolated drops.

In the illustrated example, the substantially spherical shape of the internal gel is favored by the use of a superhydrophobic surface 3 made up of a plastic polymer, for example polydimethylsiloxane (PDMS), made superhydrophobic by nano-structuring, placed on the bottom of the well.

The internal gel, in which the cells are seeded, is polymerized according to techniques known in the sector, for example by thermal variation, production of radicals by exposure to UV or visible rays depending on the type of polymer used for the production of the gel. Preferably, the gel into which the cells are seeded constitutes the internal portion of the gel-in-gel system, or internal gel, and preferably has a volume of 2-5 ?L.

On the outside of the cured inner gel, ? the external gel is deposited, with a volume preferably between 50-60 ?L, consisting of a gel having less rigidity? of the internal gel.

According to the invention, the device comprises a plurality? of channels, in a number suitable for the intended use of the device. The channels can have various shapes depending on the needs? of use of the device and the test that must be performed. For example, such channels may be straight, curved (e.g., S-shaped or serpentine), radial (e.g., radiating from, or converging toward, a central point or region, as in a V shape, U-shaped, C-shaped), elliptical, circular, square or rectangular, etc. or combinations thereof.

The channels of each well P have a preferably circular section with a diameter between 0.1-0.2 mm, preferably 0.15 mm or rectangular with dimensions of 0.1-0.5 mm x 0.1-0.2 mm, preferably 0.5 x 0.15 mm, and are independent of each other. These channels are connected to the well P in which the gel-ingel system is located, and are shaped so that one end? of each channel? in contact and flows into the side wall of the well P, constituting an entry/exit point of the well, while the other end? of the same channel ends in a small secondary cylindrical well with a diameter between 1-2 mm, preferably 1 mm, which faces the upper surface of the device, externally to a tank S. This tank S has the function of containing the growth medium of the cellular system analyzed with the device object of the invention, as described in greater detail below. In other words, each well P ? placed on the bottom of the cylindrical Tank S whose dimensions have a height h= 1.5-2.5 mm; a diameter d= 20-30 mm and a capacity? of 0.471-1.77 mL. In the illustrated example, the preferred dimensions of the tank S are: h= 2 mm and d= 26 mm with a capacity 1.06 mL in which ? place the cultivation medium, thus guaranteeing a quantity? of soil that allows you to maintain the three ?gel-in-gel? immersed in cultivation medium even for longer? days. The top of each well P ? overlooking the bottom of said tank S and ? communicating with it to receive the growth medium. More? specifically, each well P has at least one lower channel 1 which is? connected in proximity? of the bottom of the well itself and an upper channel 2 connected laterally to the well, just above the middle? of his height.

According to the invention, both channels now described are separately connected to the upper surface of the device, externally to the tank S via a respective secondary well (1? and 2?).

As stated, the lateral surface of each well P present in the device is ? connected to two independent channels 1, 2 which end respectively in two distinct points on the lateral surface of the well P, preferably opposite. A first channel 1? connected to the cockpit in its lower part (lower channel), substantially in proximity? of the bottom/base of the well P; a second channel 2? connected to the P well in its half? higher (upper channel), at a height that in the illustrated example is ? approximately 4 mm from the bottom of the well (P).

The lower channel 1 ? mainly used to wash the gel sphere containing the cells, which constitutes the internal portion of the gel-in-gel system, and therefore allows the removal of any cells not included in the sphere during the polymerization phase, this before depositing through the use of a simple micropipette the outer portion of the gel. The upper channel 2 ? instead used mainly for washing the gel-in-gel system and for changing/modifying the culture medium during the experiment.

How to ? said, the top? of each well P ? facing and communicating on the bottom of the tank S which, according to a peculiar characteristic of the invention, constitutes a reserve of growth medium common to each well P. This tank S advantageously allows experiments to be carried out even for several months. days without the need? of having to fill the wells daily to feed the cells to be analyzed present inside them, as well as? to use the same culture conditions for all three ?gel-in-gel? systems, thus allowing better reproducibility? of the experiment by reducing variables in the experiment.

Through these upper 2 and lower 1 channels, the operator can? intervene on the gel-ingel system to add or remove the culture medium, to carry out a wash or to add biochemical factors or drugs whose effects on cell migration you want to study through the use of the respective secondary wells 1? and 2? of extremity. The addition or removal of substances or media can be done by the operator manually using a syringe, a pipette or a pump or any other means suitable for adding or removing solutions, substances and liquid compositions. Alternatively, the addition or removal of substances or media can be done through mechanical means or any suitable means activated mechanically or electronically or through software, implemented according to methods known to experts in the sector.

AND? interesting to observe that when the tank S ? filled with culture medium, the P wells will share the same medium, but when the level drops to the bottom of the tank itself, the P wells become ?separated? between them.

The shape of such a tank S can? be any, as long as? has a horizontal bottom and is suitable for containing a volume of at least 1-1.5 mL of a liquid solution.

In the illustrated example, the tank S has a cylindrical shape with a diameter of 26 mm and a height of 2 mm and guarantees a continuous and large excess supply of culture medium compared to the limited volumes required by the dimensions and quantity of the tank. of cells analyzed.

Given what has been said, the presence of the S tank? particularly advantageous, as it allows to guarantee that the cells are adequately fed even for periods of a few days (e.g. the weekend) without external filling or topping up interventions and reducing hypoxia or toxicity phenomena. due to catabolites released by cells in the gel-in-gel system. This differentiates and facilitates? the use of the invention compared to other known devices.

AND? It is interesting to note that among the innovative and advantageous aspects of the device described so far, also called ?3D cell migration chip? (3D-CM chip), the following can be listed:

- Since the P wells are open towards the top, you can? have direct access to the sample with a tip.

- Depending on needs, gel spheres containing cells with small volumes, from 1 to 5 microliters, can be made using common micropipettes.

- If necessary, ? Is it possible to create and test spheres of different volumes in different P wells to analyze migrations with a shorter path? long/short; this would be impossible with a ?Kamm-type? as the volumes are strictly defined by the geometry of the channels.

- Any occlusions in the lower 1 and upper 2 channels can be removed (micro-channels).

- Can you? be used using even just some wells of the device.

- In the chip which is the object of the present invention, can it be possible? easily modify the hydrophobicity of the bottom of the well P depending on the type of gel you want to use, thanks to the fact that the hydrophobic/hydrophilic/super-hydrophobic surface 3? removable from the bottom of each well P.

- The system can? be used to carry out the polymerization process of gel-in-gel systems either through photopolymerization with exposure to UV light, or through temperature variation or with other chemical polymerization systems.

The advantages deriving from the device according to the present invention and from its use will be greater? evident to experts in the sector from the description of the examples described below.

Furthermore, the data from the experiment conducted highlighted the different capacity? migration of stem cells compared to tumor cells in the gel-in-gel system. In particular, ? It is known in the literature that adult mesenchymal stem cells such as those used by us are physiologically confined to the so-called "stem niches". In fact, it is believed that these cells are able to avoid the differentiation process during embryonic development and to colonize specific areas, with defined cytoarchitecture, which guarantee a biochemically controlled microenvironment, called niches, which would have the function of maintaining plasticity. of cells in adult life and to limit their differentiation processes [Lanza, R., 2004]. In such crypts, whose presence is? been found in numerous tissues and organs of the adult individual [Morrison, S.J., 2008], such as the endothelium, the skeletal and cardiac muscle, the liver and the cornea, these cells remain confined and relatively quiescent until they are reactivated by damage or a pathological state. In this regard, the migration experiments we conducted using the 3D-migration chip, in which there is no An invasion of the external gel by hcMSCs was observed, suggesting that this system provides an "organo-typical" model. in vitro of stem cell niches, very useful for studying the properties? physiological characteristics of the cells that constitute these niches and the identification of the factors involved in reactivation and plasticity.

EXAMPLES

Example 1: Migration of MDA-MB 231 cells in PEG-fibrinogen hydrogel detected by 3D-cell migration chip.

MDA-MB 231 tumor cells, a highly aggressive human breast adenocarcinoma line with a high capacity to invade and cross basement membranes and which do not express epithelial markers, were grown in sterile polystyrene flasks using ?Dulbecco?s modified Eagle medium? (DMEM) (Gibco, Thermo Fisher Scientific, Milan, Italy) enriched with 10% v/v fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Milan, Italy), 2 mM L-Glutamine, 100 U penicillin /mL and streptomycin 100 g/mL, at 37?C and 5% CO2. When the cells reached a confluence of 60-80% of the growth surface, they were detached, counted using a ?Thoma? cell counting chamber. with a double lattice, and resuspended at a density? of 1x10<4 >cells/?L in the precursor solution of the internal gel containing: PBS buffer (50 mM potassium phosphate, 150 mM NaCl in ddH2O) at pH 7.4, PEG-fibrinogen (8.0 mg/mL), 0.5% v/v of a PEGDA solution with a molecular weight of 10 kDa at 30% w/v and 1% v/v of a stock solution containing 10% w/v of the Irgacure photoinitiator? 2959 (Ciba Specialty Chemicals, Basel, Switzerland) in 70% ethanol (final photoinitiator concentration equal to 0.1% w/v). The ?3D-CM chip? suitably sterilized by UV treatment, ? was prepared by placing the removable sterile superhydrophobic surface, which was previously obtained by nanostructuring the PDMS polymer, into the P well.

A portion equal to 2?L of the gel precursor solution containing the MDA-MB 231 cells? was deposited with a Gilson-type micro-pipette on the superhydrophobic surfaces present in the three P wells. The entire chip was deposited. was exposed to UV light (365 nm, 4?5 mW cm<?2>) for 1 minute thus obtaining? cell microspheres. The beads were washed with PBS to remove any residual unpolymerized gel and cells not embedded in the gel using channel 1 (C1). Subsequently, the wells and the reservoir were filled with the culture medium and the ?3D-CM chip? ? was placed in a sterile petri dish in the incubator for 12 h. The next day the super-hydrophobic surfaces were removed from the wells and subsequently? the culture medium was removed to allow polymerization of the external gel. To this end ? A Gilson micro-pipette was used, drawing first from the reservoir and then from the C2 and C1 channels. After washing with PBS buffer? the external gel precursor solution consisting of PEG-fibrinogen with fibronectin (5.5 mg/mL), Irgacure 2959 photoinitiator (0.1% w/v) and PBS buffer pH 7.4 was added to a volume of 50 ?L for each well. The external gel must have a viscosity/rigidity? lower than the internal one, in order not to hinder the capacity? cell migration. The chip? was exposed to UVA irradiation (365 nm) for 5 minutes to obtain polymerization of the external gel. Subsequently, the tank and the wells were filled with the culture medium and the system was ready to begin monitoring the invasion of the external gel by the cells contained in the internal gel over time. Cellular migration/invasion? was monitored by observing the chip under an inverted bright-field microscope (Zeiss Primovert) using 4X magnification.

The experiment highlighted the ability migration of MDA-MB231 cells which, as can be seen from figure 9, is appreciable already? from the third day of cell growth.

Example 2: Migration of hcMSC stem cells in PEG-fibrinogen hydrogel detected using 3D-cell migration chip.

Lin<- >Sca-1<+ >hcMSC cells, human cardiac progenitor mesenchymal stem cells (hcMSC), extracted from the right atrial appendage of the human heart, obtained during biopsy (Buonvino S et al IJMS 2021),

were grown in sterile polystyrene flasks using ?Dulbecco?s modified Eagle medium? (DMEM) (Gibco, Thermo Fisher Scientific, Milan, Italy) enriched with 10% v/v fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Milan, Italy), 2 mM L-Glutamine, 100 U penicillin /mL and streptomycin 100 g/mL, at 37?C and 5% CO2. When the cells reached a confluence of approximately 60-80% of the growth surface, they were detached and counted using a ?Thoma? cell counting chamber. with a double lattice, and resuspended at a density? of 4x10<3 >cells/?L in the precursor solution of the internal gel containing: PBS buffer (50 mM potassium phosphate, 150 mM NaCl in ddH2O) at pH 7.4, PEG-fibrinogen (8.0 mg/mL), 0.5% v/v of a PEGDA solution with a molecular weight of 10 kDa at 30% w/v and 1% v/v of a stock solution containing 10% w/v of the Irgacure photoinitiator? 2959 (Ciba Specialty Chemicals, Basel, Switzerland) in 70% ethanol (final photoinitiator concentration equal to 0.1% w/v). The ?3D-CM chip? suitably sterilized by UV treatment, ? was prepared by placing the removable sterile superhydrophobic surface, previously obtained by nano-structuring the PDMS polymer, into well P. 5 ?L of the gel precursor solution containing the hcMSC cells were deposited with a Gilson micro-pipette on the superhydrophobic surfaces present in the three wells P. The entire chip ? was exposed to irradiation with UV light (365 nm, 4?5 mW cm<?2>) for 1 minute thus obtaining? cell microspheres. The beads were washed with PBS to remove any residual unpolymerized gel and cells not embedded in the gel using channel 1 (C1). Subsequently, the wells and the reservoir were filled with the culture medium and the ?3D cell migration chip? ? was placed in a sterile petri dish in the incubator for 12 h. The next day the super-hydrophobic surfaces were removed from the wells and ? the culture medium was removed to allow polymerization of the external gel. To this end ? A Gilson type micro-pipette was used, taking first from the reservoir and then from the C2 and C1 channels. After washing with PBS buffer? the external gel precursor solution consisting of PEG-fibrinogen with fibronectin (5.5 mg/mL), Irgacure 2959 photoinitiator (0.1% w/v) and PBS buffer pH 7.4 was added to a volume of 60 ?L for each well. The external gel must have a rigidity? (stiffness) lower than the internal one, in order not to hinder the capacity? cell migration. The chip? was exposed to the UVA lamp (365 nm) for 5 minutes to achieve polymerization of the external gel. Subsequently, the reservoir and wells were filled with the culture medium and the system was ready to begin monitoring the invasion of the external gel by the cells over time. Cellular migration/invasion? was monitored using an inverted bright-field microscope with 4X magnification (Zeiss Primovert).

The data obtained from the experimentation with adult stem cells did not detect migration/invasion of the external gel by the hcMSCs contained in the internal gel, as shown in Figure 10.

Claims (13)

1. Microfluidic device for studying in vitro cell migration on a miniaturized scale in three-dimensional systems, characterized by the fact that it provides a body of optically transparent material which includes: - one or more? wells (P) for containing a fluid, gel or similar, which constitute a gel-in-gel system in which the cells are seeded, made up of a composite matrix of natural and/or synthetic biomaterials, which creates a structure that has the capacity? to modulate the development, organization and final function of a multicellular system, a potential scaffold for cell culture; - a tank (S) to contain a culture medium which feeds said wells (P); where the top of each well (P) is? overlooking the bottom of said tank (S) and is? communicating with it to receive the growth medium; and in which each well (P) has at least one lower channel (1) which is connected in proximity? of the bottom of the well itself and at least one upper channel (2) connected laterally to the well itself, preferably just above the middle? of his height; said channels (1, 2) being separately connected with the upper surface of the device, externally to the tank (S), via a respective secondary well (1? and 2?). 2. Device according to the previous claim, characterized by the fact that on the bottom of each well (P) there is there is a removable surface (3), on which? the gel is deposited, in which said surface (3) is superhydrophobic, hydrophobic or hydrophilic, as needed. 3. Device according to one or more? of the previous claims, characterized by the fact that said gel-in-gel system is? consisting of different types of both thermo- and photopolymerizable polymers, for example agarose, gelatin, fibrin, collagen, chitosan, and/or double gelatin and/or PEG-fibrinogen systems. 4. Device according to claim 2, characterized in that, to obtain a substantially spherical shape of the internal gel, said removable surface (3) located on the bottom of the well (P) is of a superhydrophobic character, made of a plastic polymer, for example polydimethylsiloxane (PDMS), made superhydrophobic by nano-structuring. 5. Device according to one or more? of the previous claims, characterized by the fact that the internal portion of the gel-in-gel system is? consists of an internal gel, in which the cells are trapped, while on the outside of the polymerized internal gel, it is an external gel is deposited, made of the same material as the non-polymerized internal gel. 6. Device according to one or more? of the previous claims, characterized by the fact that the channels (1, 2) of each well (P) have a circular section with a diameter between 0.1-0.2 mm, and preferably equal to 0.15 mm or a rectangular section with dimensions ranging between 0.1-0.5mm in height and 0.1-0.2mm in width, and preferably equal to 0.5 x 0.15mm, and are independent of each other. 7. Device according to one or more? of the previous claims, characterized by the fact that the channels (1, 2) of each well (P) are connected to the well (P) in which the gel-in-gel system is located, and are shaped so that a extremity? of each channel? in contact and flows into the side wall of the well (P) constituting an entry/exit point of the well, while the other end? of the same channel ends in the respective secondary well (1?, 2?) which faces the upper surface of the device, outside the tank (S). 8. Device according to one or more? of the previous claims, characterized by the fact that each well (P) present on the bottom of the tank (S) has a substantially cylindrical shape with a height h = 4-6 mm, a diameter d = 4-6 mm and an internal volume V = 50 -169 ?l; preferably with a height of 5 mm and a diameter of 5 mm and an internal volume of 98.2 ?l. 9. Device according to one or more? of the previous claims, characterized by the fact that said tank (S) in which is? put the cultivation medium, ? substantially cylindrical and has a height h= 1.5-2.5 mm and a diameter d= 20-30 mm; preferably a height h= 2 mm and a diameter d= 26 mm with a capacity? 1.06 mL, thus guaranteeing a quantity? of soil that allows you to maintain the three ?gel-in-gel? immersed in cultivation medium even for longer? days. 10. Device according to one or more? of the previous claims, characterized by the fact that the lateral surface of each well (P) present in the device is? connected to said two independent channels (1, 2) which end respectively in two distinct points on the lateral surface of the well (P), preferably opposite. 11. Device according to one or more? of the previous claims, characterized by the fact that the lower channel (1) is usable to wash the gel sphere containing the cells, which constitutes the internal portion of the gel-ingel system, and therefore to remove any cells not included in the sphere during the polymerization phase, before depositing the external portion of the gel using a simple micropipette; and by the fact that the upper channel (2) ? usable for washing the gel-gel system and for changing/modifying the culture medium during the experiment. 12. Device according to one or more? of the previous claims, characterized by the fact that said upper (2) and lower (1) channels of each well (P) are configured to allow an operator to intervene on the gel-ingel system to add or remove the culture medium, to perform a wash or to add biochemical factors or drugs whose effects on cell migration are to be studied, using the respective secondary wells (1? and 2?) at the ends. 13. Device according to one or more? of the previous claims, characterized by the fact that said tank (S) has a horizontal bottom and has a shape suitable for containing a volume of at least 1-1.5 mL of a liquid solution.