New microfluidic device and method for its manufacture

Abstract

The present invention relates to a new microfluidic device for in vitro cell culture and drug screening, and methods for its manufacture The present invention also relates to the uses of the microfluidic device of the invention and to the methods which it enables to implement.

Description

[0001] NEW MICROFLUIDIC DEVICE AND METHOD FOR ITS MANUFACTURE FIELD OF THE INVENTION

[0002] The present invention relates to a new microfluidic device for in vitro cell culture and drug screening, and methods for its manufacture. The present invention also relates to the uses of the microfluidic device of the invention and to the methods which it enables to implement.

[0003] PRIOR ART

[0004] For years, cell cultures have been conducted in traditional formats like flasks, Petri dishes, and bioreactors, but these methods struggle to replicate the dynamic cell-environment interactions that influence cellular functions and responses. Conventional processes often involve static conditions, lack real-time monitoring, and introduce unwanted gradients, leading to cellular stress and high costs. Microfluidic devices with 3D cell structures offer a solution by enabling precise control over the microenvironment, improving the biological relevance of cell models while maintaining or enhancing experimental throughput. The 3D cell culture systems can be categorized into two segments: 3D scaffold-based and scaffold-free systems (Stefano et al. “The impact of microfluidics in high-throughput drug-screening applications.” Biomicrofluidics 16.3 (2022)). In the scaffold-based system, cells are seeded on pre-fabricated scaffolds in an extracellular matrix. The seeded cells take the scaffolds as support to adhere, grow, migrate, and differentiate. Unlike in scaffold-free systems, cells are allowed to migrate, differentiate, and reassemble to form spheroids or organoids without any structural support. The scaffold-free system recapitulates the environment of 3D cell-cell interactions. Among many scaffold-free systems, the hanging drop method and microfluidics-based methods (phase guide systems, pillar-based designs, membrane-based culture) are the most promising ones.

[0005] Hanging drop method. The hanging drop method leverages surface tension to suspend a culture media droplet containing cells, enabling spheroid formation. Tung etal. (“High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array.” Analyst 136.3 (2011): 473-478) developed a 384-well hanging drop plate with an access hole for cell seeding and a media exchange system to minimize evaporation. Marimuthu et al. (“Multi-size spheroid formation using microfluidic funnels.” Lab on a Chip 18.2 (2018): 304-314) further demonstrated that cone-shaped wells allow the formation of multi-size spheroids from single-cell loading inlets. While these systems are cost-effective, simple, and suitable for high-throughput screening without scaffolds, they face challenges related to handling, nutrient gradients, lack of perfusion, and evaporation.

[0006] Microfluidics-based system'. Alternative strategies involve microfluidics-based 3D cell culture methods, including pillar-guided (Toh, Yi-Chin, et al. “A microfluidic 3D hepatocyte chip for drug toxicity testing.” Lab on a Chip 9.14 (2009): 2026-2035), phase-guided (Trietsch, Sebastiaan J., etal. “Microfluidic title plate for stratified 3D cell culture.” Lab on a Chip 13.18 (2013): 3548-3554), and membrane-based devices (Vatine, Gad D., et al. “Human iPSC-derived blood-brain barrier chips enable disease modeling and personalized medicine applications.” Cell stem cell 24.6 (2019): 995-1005). In both pillar-guided and phase-guided systems, a cell-laden hydrogel is injected and directed into a predefined area within a microfluidic channel network. Pillar structures or horizontal phase boundaries confine the liquid, while other channels facilitate media or drug exchange. Similarly, membrane-based systems use porous membranes to separate channels, allowing the flow of cell-laden solutions to form cell layers on the membrane. The top and bottom channels serve as media or drug delivery pathways, supporting cell proliferation in a 3D environment.

[0007] The scaffold-free systems can better replicate the cellular environment, but they still face challenges such as perfusion, shear stress range, throughput, cell recovery, and the need for external flow equipment.

[0008] BRIEF DESCRISPTION

[0009] In order to overcome these drawbacks, the inventors have conceived a new microfluidic device (4) for in vitro cell culture and drug screening wherein a 3D pattern wetting is implemented so as to trap a hydrogel.

[0010] A first aim of the invention is therefore the use of hydrophilicity difference (wettability contrast) to implement a 3D pattern wetting so as to trap a liquid hydrogel precursor solution (12) liable to be in situ polymerized within a microfluidic device (4).A second and third aims of the invention concern said microfluidic device (4) and processes of manufacture of the microfluidic device (4).

[0011] Further aims of the invention also relate to uses of the microfluidic device (4) of the invention and to the methods which it enables to implement.

[0012] DETAILED DESCRIPTION

[0013] According to a first aspect, the subject matter of the invention relates to the use of a hydrophilicity difference to implement a 3D pattern wetting so as to trap a liquid hydrogel precursor solution (12) liable to be in situ polymerized within a microfluidic device (4), said microfluidic device (4) comprising at least three layers (1, 2, 3):

[0014] - a top layer (1) containing hollow spaces;

[0015] - a microfluidic middle layer (2) containing hollow spaces; and

[0016] - a bottom layer (3),

[0017] the stacking of these three layers (1, 2, 3) defining at least one inlet (7) adjacent to a chamber (8), said chamber (8) forming a trapping zone for said liquid hydrogel precursor solution (12) liable to be in situ polymerized,

[0018] said chamber (8) comprising:

[0019] - a lower face [which corresponds to the top face of said bottom layer 3 ] - a top face [which corresponds to the bottom face of said top layer (7)]; and - 4 sides (8a, 8b, 8c, 8d), the side (8a) being opposed to the side (8b) and the side (8c) being opposed to the side (8d),

[0020] said chamber (8) being:

[0021] - opened on 2 opposite sides (8a, 8b), the side (8a) being adjacent to said at least one inlet (7);

[0022] - in direct fluid communication with said at least one inlet (7) through said side (8a); and

[0023] - located in layer (2) and between layers (1) and (3),

[0024] wherein at least the surface of the lower face of said chamber (8) is more hydrophilic than the surface of said inlet (7), said hydrophilicity being measured by contact angle method (see Figure 13).“Hydrophilicity difference” refers to the fact that chamber (8) forming a trapping zone is more hydrophilic than the surface of said inlet (7). It means that the invention allows, when a liquid hydrogel precursor solution (12) is loaded into the inlet (7), for it to be immediately directed and trapped in the chamber (8) because it is the ideal environment for said liquid hydrogel precursor solution (12). In other words, the invention corresponds to a microfluidic device (4) enabling the suction and imprisonment of a liquid hydrogel precursor solution (12) liable to be in situ polymerized in a chamber (8) using a 3D pattern wetting within a microfluidic device (4). “3D pattern wetting” refers to trapping liquid / hydrogel using the combination of geometry and wettability. The implementation of said 3D pattern wetting is directly linked to the manufacture of specific hydrophilic spots (11, optionally 11’) and / or hydrophobic spot (18). One hydrophilic spot (11) is placed at the lower surface of said chamber (8) and / or one hydrophobic spot (18) is placed at the lower surface of said inlet (7), and optionally one hydrophilic spot (11 ’) is placed at the top surface of said chamber (8). Once said liquid hydrogel precursor solution (12) is located within said chamber (8), it is cured. The hydrogel (12a) obtained is therefore supplied with liquid (e.g. culture medium) by means of the inlet (7) and outlet (5) which surround said chamber (8), the both sides (8a, 8b) of which are opened, and with which it is in fluid communication.

[0025] As mentioned, hydrophilicity is measured by contact angle method which is known in the art. Static contact angle measurement which is the most common method that utilizes the sessile drop method. A contact angle goniometer and a high-resolution camera are used to capture an image of the drop sitting on the surface from the side view and measure the contact angle. Alternatively, the dynamic contact angle can be measured. Dynamic contact angles are presented as receding and advancing angles, depending on the direction where the drop of liquid is moving on a tilted surface. The angle of contact where the drop begins its movement is referred to as the roll-off angle.

[0026] According to another embodiment, the subject matter of the invention relates to the use as described above of a hydrophilicity difference, wherein:

[0027] - at least a first hydrophilic spot (11) is placed at the lower surface of said chamber (8) [which corresponds to the top of the layer (3)] and extends contiguously towards the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to form an additional hydrophilic surface (Ila) to promote thesuction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0028] said first hydrophilic spot (11):

[0029] ■ being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2- hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0030] ■ being etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) [which corresponds to the top of the layer 3 ] and / or

[0031] - a hydrophobic spot (18) is placed at the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to promote the confinement of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0032] said hydrophobic spot (18):

[0033] ■ being made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; or

[0034] ■ being a superhydrophobic surface, structured with micro / nano- texturization on the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] (see Figure 13).

[0035] According to another embodiment, the subject matter of the invention relates to the use as described above of a hydrophilicity difference, wherein at least a first hydrophilic spot (11) is placed at the lower surface of said chamber (8) [which corresponds to the top of the layer (3)] and extends contiguously towards the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0036] said first hydrophilic spot (11):

[0037] ■ being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0038] ■ being etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) [which corresponds to the top of the layer (3)] see Figure 13 - Coating design 2).

[0039] According to another embodiment, the subject matter of the invention relates to the use as described above of a hydrophilicity difference, wherein a hydrophobic spot (18) is placed at the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to promote the confinement of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0040] said hydrophobic spot (18):

[0041] ■ being made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; or

[0042] ■ being a superhydrophobic surface, structured with micro / nano- texturization on the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] (see Figure 13 - Coating design 3).

[0043] According to another embodiment, the subject matter of the invention relates to the use as described above of a hydrophilicity difference, wherein:

[0044] - at least a first hydrophilic spot (11) is placed at the lower surface of said chamber (8) [which corresponds to the top of the layer (3)] and extends contiguously towards the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0045] said first hydrophilic spot (11):

[0046] ■ being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2- hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0047] ■ being etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) [which corresponds to the top of the layer 3 ]and

[0048] - a hydrophobic spot (18) is placed at the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to promote the confinement of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0049] said hydrophobic spot (18):

[0050] ■ being made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; or

[0051] ■ being a superhydrophobic surface, structured with micro / nano- texturization on the lower surface of said inlet (7) [which corresponds to the top of the layer (3)].

[0052] According to another embodiment, the subject matter of the invention relates to the use as described above of a hydrophilicity difference, wherein a second hydrophilic spot (11’) is placed at the top surface of said chamber (8) [which corresponds to the bottom of the layer (l \, said second hydrophilic spot (IT):

[0053] - being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0054] - being etched surfaces structured with a laser or a plasma on the top surface of said chamber (8) [which corresponds to the bottom of the layer (lf\ (see Figure 13 - Coating design 1).

[0055] According to another embodiment, the subject matter of the invention relates to the use as described above of a hydrophilicity difference, wherein said microfluidic device (4) further comprises a third hydrophilic spot (101) placed at the surface of said side (8c) and a fourth hydrophilic spot (1001) placed at the surface of said side (8d),

[0056] said third hydrophilic spot (101) and said fourth hydrophilic spot (1001):

[0057] - being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethylmethacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0058] - being etched surfaces structured with a laser or a plasma on the surfaces of said sides (8c, 8d) (see Figure 17).

[0059] According to another embodiment, the subject matter of the invention relates to the use as described above of at least a first and a second hydrophilic spots (11, 11’) to implement a 3D pattern wetting so as to trap a liquid hydrogel precursor solution (12) liable to be in situ polymerized within a microfluidic device (4),

[0060] said microfluidic device (4) comprising at least three layers (1, 2, 3):

[0061] - a top layer (1) containing hollow spaces;

[0062] - a microfluidic middle layer (2) containing hollow spaces; and

[0063] - a bottom layer (3),

[0064] the stacking of these three layers (1, 2, 3) defining at least one inlet (7) adjacent to a chamber (8), said chamber (8) forming a trapping zone for said liquid hydrogel precursor solution (12) liable to be in situ polymerized,

[0065] said chamber (8) comprising:

[0066] - a lower face [which corresponds to the top face of said bottom layer 3 ] - a top face [which corresponds to the bottom face of said top layer (7)]; and - 4 sides (8a, 8b, 8c, 8d), the side (8a) being opposed to the side (8b) and the side (8c) being opposed to the side (8d),

[0067] said chamber (8) being:

[0068] - opened on 2 opposite sides (8a, 8b), the side (8a) being adjacent to said at least one inlet (7);

[0069] - in direct fluid communication with said at least one inlet (7) through said side (8a); and

[0070] - located in layer (2) and between layers (1) and (3),

[0071] wherein:

[0072] - said first hydrophilic spot (11) is placed at the lower surface of said chamber (8) [which corresponds to the top of the layer (3)] and extends contiguously towards the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8);- said second hydrophilic spot (11’) is placed at the top surface of said chamber (8) [which corresponds to the bottom of the layer (7)]; and

[0073] - said first and second hydrophilic spots (11, 11’):

[0074] ■ are made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), desiccated (dried) cell culture medium (e.g. DMEM, MEM, RPMI, F-12, OPTI-MEM, MCCOY’S 5 A MEDIUM, KSFM, etc.) or a mixture thereof; or

[0075] ■ are etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) [which corresponds to the top of the layer (3)] and the top surface of said chamber (8) [which corresponds to the bottom of the layer (1)} (see Figure 13 - Coating design 1).

[0076] “Chamber (8)” refers to the compartment resulting from the stacking of at least three layers (1, 2, 3) and which houses the liquid hydrogel precursor solution (12) liable to be in situ polymerized or the hydrogel (12a) when it is cured. In particular, the chamber base is a trapezoid or a parallelogram (such as a rectangle or a square). Advantageously, the chamber base is trapezoidal, and its dimensions are 1.5 mm x 0.5 mm, 1.5 mm (parallel side 1, parallel side 2, distance between parallel sides respectively). In particular, the chamber (8) has a height of 0.25 mm, which is also the height of the microfluidic middle layer (2).

[0077] “Hydrophilic spots (11, 11’)” refer to hydrophilic patches / surf aces located at the base (plane 3’ of layer (3)) and the top (plane 1 of the layer (1)) of the chamber (8) and which trap a liquid hydrogel precursor solution (12) liable to be in situ polymerized. The hydrophilic properties of said hydrophilic spots (11, 11’) allow the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from an inlet (7) to said chamber (8). To promote said suction, the base (plane 3’ of layer (3)) of the chamber (8) has an additional hydrophilic surface (Ila = extra edge) (1.5 mm x 0.1 - 0.25 mm; length, width respectively located at the base of said inlet (7). To manufacture hydrophilic spots (11, 11’), either a coating with hydrophilic materials is done or etched surfaces (rough surfaces) using a laser or a plasma are engraved. Consequently, it is understood that the hydrophilic properties of said hydrophilic spots (11, 11’) come from either the nature of the coated material on said hydrophilic spots (11, 11’), or the mechanical structure / pattern designed or engraved on said hydrophilic spots (11, 11’).Interestingly, these hydrophilic spots (11, optionally 11’) can be used alone or combined with the hydrophobic spot (18) described elsewhere to optimize droplet printing and trapping.

[0078] According to another embodiment, the subject matter of the invention relates to the use as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) made of poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), desiccated (dried) cell culture medium (e.g. DMEM, MEM, RPMI, F-12, OPTI-MEM, MCCOY’S 5 A MEDIUM, KSFM, etc.) or a mixture thereof “Mixture thereof’ refers to PEG / PEO, PEG / PVA, PEG / Desiccated cell culture medium, PEO / PVA, PEO / Desiccated cell culture medium, PVA / Desiccated cell culture medium, PEG / PEO / PVA, PEG / PEO / Desiccated cell culture medium, PEG / PVA / Desiccated cell culture medium, PEO / PVA / Desiccated cell culture medium and PEG / PEO / PVA / Desiccated cell culture medium.

[0079] According to another embodiment, the subject matter of the invention relates to the use as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) made of poly(ethylene glycol) (PEG), polyethylene oxide) (PEO)or polyvinyl alcohol (PVA). According to another embodiment, the subject matter of the invention relates to the use as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) made of desiccated (dried) cell culture medium (e.g. DMEM, MEM, RPMI, F-12, OPTI-MEM, MCCOY’S 5 A MEDIUM, KSFM, etc.). According to another embodiment, the subject matter of the invention relates to the use as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) made of PEG / PEO, PEG / PVA, PEG / Desiccated cell culture medium, PEO / PVA, PEO / Desiccated cell culture medium, PVA / Desiccated cell culture medium, PEG / PEO / PVA, PEG / PEO / Desiccated cell culture medium, PEG / PVA / Desiccated cell culture medium, PEO / PVA / Desiccated cell culture medium or PEG / PEO / PVA / Desiccated cell culture medium

[0080] According to another embodiment, the subject matter of the invention relates to the use as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) and the top surface of said chamber (8). “Etched surfaces structured with a laser or a plasma” refer to rough surfaces, the patterning thermoplastic hydrophilicity of which is made using a laser or a plasma. This technique creates asperities in the thermoplastic used to manufacture layers of the microfluidic device (4) of the invention so as to create angles. This engraved structure thenpresents asperities and “micro” receptacles which allow the liquid hydrogel precursor solution (12) to be sucked up in this mechanically hydrophilic pattern. This technique is well documented. See for instance:

[0081] ZILIO, Caterina, SOLA, Laura, DAMIN, Francesco, et al. Universal hydrophilic coating of thermoplastic polymers currently used in microfluidics. Biomedical microdevices, 2014, vol.

[0082] 16, p. 107-114. (topaz 8007 can be made hydrophilic using plasma);

[0083] SINGH, Sanasam Sunderlal and SAMUEL, GL Near-infrared femtosecond laser direct writing of microchannel and controlled surface wettability. Optics & Laser Technology, 2024, vol. 170, p. 110214. (PMMA surface can be modified to hydrophilic);

[0084] QI, Heng, CHEN, Tao, YAO, Liying, et al. Hydrophilicity modification of poly (methyl methacrylate) by excimer laser ablation and irradiation. Microfluidics and Nanofluidics, 2008, vol. 5, p. 139-143.; and

[0085] VARGAS, Matheus JT, NfEUWOUDT, Michel K., ARUL, Rakesh, et al. Direct laser writing of hydrophobic and hydrophilic valves in the same material applied to centrifugal microfluidics. RSC advances, 2023, vol. 13, no. 32, p. 22302-22314. (polycarbonate hydrophilicity).

[0086] Interestingly, said first and optionally second hydrophilic spots (11, 11’), when they correspond to etched surfaces structured with a laser or a plasma they could be directly engraved on the base (plane 3’) of layer (3) and the top (plane 1) of the layer (1) of the chamber (8), respectively; or

[0087] they could be designed on other thermoplastic elements and then glued on the base (plane 3’) of layer (3) and the top (plane 1) of the layer (1) of the chamber (8).

[0088] According to another embodiment, the subject matter of the invention relates to the use as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) etched surfaces structured with a laser on the lower surface of said chamber (8) and the top surface of said chamber (8).

[0089] “Hydrophobic spot (18)” refers to hydrophobic patches / surfaces located at the base (plane 3’ of layer (3)) of the inlet (7). The hydrophobic spot (18) has a continuous hydrophobic layer so that the wettability contrast between the chamber (8) interior and the hydrophobic surroundings promotes confinement of the liquid hydrogel precursor solution (12) within chamber (8) through capillary and contact-angle difference or wettability contrast. Possible hydrophobic treatments include but are not limited to: vapor-phase silanization with perfluoroalkylsilanes (for example trichloro / perfhioroalkyl silanes), deposition of fluoropolymer coatings (e.g.,Teflon™-like films, CYTOP®, AF-type coatings), parylene coating followed by fluorination, plasma polymerization to deposit hydrophobic films, or spray / surface coatings of commercially available hydrophobic agents. Alternatively, micro / nano-texturing (for example laser ablation followed by a fluorosilane treatment) may be used to create superhydrophobic regions. When using any of these treatments, masking of the chamber interior or selective deposition may be performed so that the chamber interior remains sufficiently wettable for capillary uptake.

[0090] Interestingly, this hydrophobic spot (18) can be used alone or combined with the hydrophilic spots (11, optionally 11’) described elsewhere to optimize droplet printing and trapping.

[0091] According to another embodiment, the subject matter of the invention relates to the use as described above, wherein said hydrophobic spot (18):

[0092] ■ is made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; or

[0093] ■ is a superhydrophobic surface, structured with micro / nano-texturization on the lower surface of said inlet (7).

[0094] According to another embodiment, the subject matter of the invention relates to the use as described above, wherein said hydrophobic spot (18) is made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer. According to another embodiment, the subject matter of the invention relates to the use as described above, wherein said hydrophobic spot (18) is a superhydrophobic surface, structured with micro / nano-texturization on the lower surface of said inlet (7).

[0095] According to a second aspect, the subject matter of the invention relates to a microfluidic device (4) made up of a stack of at least three layers (1, 2, 3):

[0096] a top layer (1) containing hollow spaces;

[0097] a microfluidic middle layer (2) containing hollow spaces; and

[0098] a bottom layer (3)

[0099] the stacking of these three layers (1, 2, 3) defining at least one inlet (7) adjacent to a chamber (8), said chamber (8) forming a trapping zone for a liquid hydrogel precursor solution (12) liable to be in situ polymerized,

[0100] said chamber (8) comprising:

[0101] - a lower face [which corresponds to the top face of said bottom layer 3 ]- a top face [which corresponds to the bottom face of said top layer (7)]; and - 4 sides (8a, 8b, 8c, 8d), the side (8a) being opposed to the side (8b) and the side (8c) being opposed to the side (8d),

[0102] said chamber (8) being:

[0103] - opened on 2 opposite sides (8a, 8b), the side (8a) being adjacent to said at least one inlet (7);

[0104] - in direct fluid communication with said at least one inlet (7) through said side (8a); and

[0105] - located in layer (2) and between layers (1) and (3),

[0106] wherein at least the surface of the lower face of said chamber (8) is more hydrophilic than the surface of said at least one inlet (7), said hydrophilicity being measured by contact angle method.

[0107] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) further containing at least:

[0108] an outlet (5) located in said layers (1) and (2); and

[0109] a well (6) located in said layers (1) and (2) which comprises said at least inlet (7) and said chamber (8),

[0110] said chamber (8) optionally comprising an injection port (13),

[0111] said outlet (5) and said chamber (8) being in fluid communication through the side (8b) of the chamber (8).

[0112] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) comprising:

[0113] - at least a first hydrophilic spot (11) placed at the lower surface of said chamber (8) [which corresponds to the top of the layer (3)], said at least first hydrophilic spot (11) extending contiguously towards the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8), said first hydrophilic spot (11) which form a 3D pattern wetting:

[0114] ■ being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0115] ■ being etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) [which corresponds to the top of the layer 3 ] and / or

[0116] - a hydrophobic spot (18) placed at the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to promote the confinement of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0117] said hydrophobic spot (18):

[0118] ■ being made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; or

[0119] ■ being a superhydrophobic surface, structured with micro / nano- texturization on the lower surface of said inlet (7) [which corresponds to the top of the layer (3)].

[0120] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) comprising at least a first hydrophilic spot (11) placed at the lower surface of said chamber (8) [which corresponds to the top of the layer (3)], said at least first hydrophilic spot (11) extending contiguously towards the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0121] said first hydrophilic spot (11) which form a 3D pattern wetting:

[0122] ■ being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2- hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0123] ■ being etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) [which corresponds to the top of the layer (3)].

[0124] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) comprising a hydrophobic spot (18)placed at the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to promote the confinement of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0125] said hydrophobic spot (18):

[0126] ■ being made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; or

[0127] ■ being a superhydrophobic surface, structured with micro / nano- texturization on the lower surface of said inlet (7) [which corresponds to the top of the layer (3)].

[0128] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) comprising:

[0129] - at least a first hydrophilic spot (11) placed at the lower surface of said chamber (8) [which corresponds to the top of the layer (3)], said at least first hydrophilic spot (11) extending contiguously towards the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8), said first hydrophilic spot (11) which form a 3D pattern wetting:

[0130] ■ being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2- hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0131] ■ being etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) [which corresponds to the top of the layer 3 ] and

[0132] - a hydrophobic spot (18) placed at the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to promote the confinement of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0133] said hydrophobic spot (18):

[0134] ■ being made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; orbeing a superhydrophobic surface, structured with micro / nano- texturization on the lower surface of said inlet (7) [which corresponds to the top of the layer (3)].

[0135] The expression “wherein at least the surface of the lower face of said chamber (8) is more hydrophilic than the surface of said at least one inlet (7)” refers to the fact that other surfaces of said chamber (8) may be more hydrophilic than the surface of said at least one inlet (7). For instance and as described hereafter, a second hydrophilic spot (IE) may be placed at the top surface of said chamber (8), a third hydrophilic spot (101) may be placed at the surface of said side (8c) and / or a fourth hydrophilic spot (1001) may be placed at the surface of said side (8d). Interestingly, it has to be pointed out that said hydrophilic spots cover 100% of the area of interest. It means that:

[0136] ■ said first hydrophilic spot (11) covers 100% of the surface of the lower face of said chamber (8);

[0137] ■ said second hydrophilic spot (11’), when it presents, covers 100% of the surface of the top face of said chamber (8);

[0138] ■ said third hydrophilic spot (101), when it presents, covers 100% of the surface of the side (8c) of said chamber (8); and

[0139] ■ said fourth hydrophilic spot (1001), when it presents, covers 100% of the surface of the side (8d) of said chamber (8).

[0140] According to another embodiment, the subject matter of the invention relates thus to the microfluidic device (4) as described above, said microfluidic device (4) further comprising a second hydrophilic spot (11’) placed at the top surface of said chamber (8) [which corresponds to the bottom of the layer (l \,

[0141] said second hydrophilic spot (IT):

[0142] - being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0143] - being etched surfaces structured with a laser or a plasma on the top surface of said chamber (8) [which corresponds to the bottom of the layer (l \.According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above made up of a stack of at least three layers (1, 2, 3): a top layer (1), a microfluidic middle layer (2) and a bottom layer (3),

[0144] said microfluidic device (4) containing at least:

[0145] an outlet (5) located in layers (1) and (2); and

[0146] a well (6) located in layers (1) and (2) which comprises an inlet (7) and a chamber (8) wherein:

[0147] ■ said inlet (7) is located in layers (1) and (2), and is adjacent to a side (8a) of a chamber (8);

[0148] ■ said chamber (8) forming a trapping zone for a liquid hydrogel precursor solution (12) liable to be in situ polymerized is opened on 2 opposite sides (8a, 8b) and is located in layer (2) and between layers (1) and (3), said chamber (8) optionally comprising an injection port (13); and ■ said inlet (7) and said chamber (8) are in direct fluid communication through the side (8a) of the chamber (8),

[0149] said outlet (5) and said chamber (8) being in fluid communication through the side (8b) of the chamber (8),

[0150] wherein:

[0151] - the lower surface of said chamber (8) [which corresponds to the top of the layer (3)] comprises a first hydrophilic spot (11), said first hydrophilic spot (11) extends contiguously towards the lower surface of said inlet (7) [which corresponds to the top of the layer (3)] so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8); and

[0152] - the top surface of said chamber (8) [which corresponds to the bottom of the layer (1)} comprises a second hydrophilic spot (11’),

[0153] said first and second hydrophilic spots (11, 11’) which form a 3D pattern wetting:

[0154] - being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), desiccated (dried) cell culture medium (e.g. DMEM, MEM, RPMI, F-12, OPTI- MEM, MCCOY’S 5 A MEDIUM, KSFM, etc.) or a mixture thereof; or

[0155] - are etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) [which corresponds to the top of the layer (3)] and the top surface ofsaid chamber (8) (which corresponds to the bottom of the layer (1)~\ (see Figure 1).

[0156] Interestingly, the microfluidic device (4) addresses a few salient problems:

[0157] 1. Using the unique capillary -based liquid trapping mechanism, the microfluidic device (4) can handle very small volumes in the pL range or 100 nL even (biological and chemical materials). This enables testing on primary patient cells as well as any cells of high value such as differentiated cells or cell lines and reduces the cost of the operation.

[0158] 2. The microfluidic device (4) can be integrated with droplet dispensers / printers. This makes it possible to automate the hydrogel and media / drugs seeding process without using complicated pumping and tubing setups. Everything can be automated in this innovation, from hydrogel injection to sealing the well plates.

[0159] 3. The microfluidic device (4) has a hydrostatic-based flow technique with no added external pumps or tubings.

[0160] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said chamber (8) has a trapezoidal shape, the dimensions of which are 1.5 mm x 0.5 mm, 1.5 mm, 0.25 mm (parallel side 1, parallel side length 2, distance between parallel sides respectively).

[0161] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein the dimensions of said additional hydrophilic surface (Ila) are 1.5 mm x 0.1 - 0.25 mm (length, width respectively).

[0162] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) made of poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), desiccated (dried) cell culture medium (e.g. DMEM, MEM, RPMI, F-12, OPTI-MEM, MCCOY’S 5 A MEDIUM, KSFM, etc.) or a mixture thereof.

[0163] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) made of poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO)or polyvinyl alcohol (PVA). According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) made of desiccated (dried) cell culture medium (e.g. DMEM,MEM, RPMI, F-12, OPTI-MEM, MCCOY’S 5 A MEDIUM, KSFM, efc ). According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) made of PEG / PEO, PEG / PVA, PEG / Desiccated cell culture medium, PEO / PVA, PEO / Desiccated cell culture medium, PVA / Desiccated cell culture medium, PEG / PEO / PVA, PEG / PEO / Desiccated cell culture medium, PEG / PVA / Desiccated cell culture medium, PEO / PVA / Desiccated cell culture medium or PEG / PEO / PVA / Desiccated cell culture medium

[0164] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said first and optionally second hydrophilic spots (11, 11’) is (are) etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8) and the top surface of said chamber (8). In particular, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said first and second hydrophilic spots (11, 11’) are etched surfaces structured with a laser on the lower surface of said chamber (8) and the top surface of said chamber (8).

[0165] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said first hydrophilic spot (11) being placed at the lower surface of said chamber (8) and / or said second hydrophilic spot (11’) placed at the top surface of said chamber (8) is(are) arranged in a 50-to-100-micron depth cavity(ies) engraved inside said lower surface of said chamber (8) and / or said top surface of said chamber (8) (see Figure 14)

[0166] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) further comprising a third hydrophilic spot (101) placed at the surface of said side (8c) and a fourth hydrophilic spot (1001) placed at the surface of said side (8d),

[0167] said third hydrophilic spot (101) and said fourth hydrophilic spot (1001):

[0168] - being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0169] - being etched surfaces structured with a laser or a plasma on the surfaces of said sides (8c, 8d) (see Figure 17).Interestingly, in this configuration, no physical flow gap may initially be present. Instead, the flow pathway is generated dynamically during operation. A hydrophilic spot is applied in such a manner that, upon contact with aqueous culture medium, the hydrophilic spot gradually dissolves, thereby creating a vacant space that subsequently serves as a flow channel for the culture medium. The hydrophilic spots may be tuned to control the rate and extent of space formation required for medium perfusion. Materials used for generating the flow space may include water-soluble coatings such as polyethylene glycol (PEG) or similar polymers, which dissolve upon exposure to aqueous media including cell culture medium. Other surfaces of the chamber (8) may be coated with materials that do not dissolve in aqueous media or that form stable hydrogel layers, such as poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), or similar polymers. By selectively applying different coating materials to specific faces / sides of the chamber (8), the formation of flow spaces and hydrogel confinement can be controlled according to experimental requirements. Through this approach, the microfluidic device (4) as described above enables programmable formation of perfusion pathways while maintaining hydrogel stability within the chamber region.

[0170] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said chamber (8) comprises an injection port (13). “Injection port” refers to a hemispherical cut-out located in the top layer (1) that facilitates the injection process of said liquid hydrogel precursor solution (12) liable to be in situ polymerized (or cured).

[0171] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said outlet (5) and said chamber (8) are in fluid communication through the side (8b) of the chamber (8) by a channel (10) located in layer (2) (see Figure 2, 5-6). “Channel (10)” refers to a hollow micropipe with an inlet and an outlet, or vice versa, through which a fluid can flow. In particular, the width of said channel (10) is 0.2 mm.

[0172] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said outlet (5) comprises a cured porous polymer network layer (50),

[0173] in particular said cured porous polymer network layer (50) being made of chitosan, polyethylene glycol diacrylate (PEGDA), polyethylene glycol monomethacrylate (PEGMA), porous polydimethylsiloxane (porous PDMS), Cellulose, polyvinylidene difluoride (PVDF),Polycarbonate (PC), Polysulfone (PS), Polyethersulfone (PES), Nylon, PTFE membranes or other air-permeable polymers with minimal shrinkage upon curing (see Figure 15).

[0174] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said outlet (5) comprises a cured porous polymer network layer (50), said cured porous polymer network layer (50) being made of chitosan, polyethylene glycol diacrylate (PEGDA), polyethylene glycol monomethacrylate (PEGMA), porous polydimethylsiloxane (porous PDMS), Cellulose, polyvinylidene difluoride (PVDF), Polycarbonate (PC), Polysulfone (PS), Polyethersulfone (PES), Nylon, PTFE membranes or other air-permeable polymers with minimal shrinkage upon curing.

[0175] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said microfluidic device (4) further comprises an inlet (9) located in layers (1) and (2),

[0176] said outlet (5), said chamber (8) and said inlet (9) being in fluid communication through a channel (10) located in layer (2), said channel (10) being connected to said chamber (8) through the side (8b) of the chamber (8) (see Figures 3-4).

[0177] “Fluid communication” refers to the fact that a liquid can flow between at least two distinct components of the microfluidic device (4) of the invention with the proviso that these two elements have necessary means for the circulation of said liquid. In other words, if:

[0178] ■ an inlet (7) is in fluid communication with a chamber (8), it means a liquid can flow between said inlet (7) and said chamber (8) (or a liquid can flow from said inlet (7) to said chamber (8));

[0179] ■ a chamber (8) is in fluid communication with an outlet (5), it means a liquid can flow between said chamber (8) and said outlet (5) (or a liquid can flow from said chamber (8) to said outlet (5));

[0180] ■ a chamber (8) is in fluid communication with an outlet (5) and a channel (10), it means a liquid can flow between said chamber (8), said channel (10) and said outlet (5) (or a liquid can flow from said chamber (8) to said outlet (5) by said channel (10)); or

[0181] ■ an inlet (9) is in fluid communication with a channel (10), a chamber (8) and an outlet (5), it means a liquid can flow between said inlet (9), said channel (10), said chamber (8) and said outlet (5) (or a liquid can flow from said inlet (9) to said outlet (5) by said channel (10) and said chamber (8)).According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said chamber (8) being divided into two adjacent compartments (14, 14’), aligned with the axis made from opposite sides (8a, 8b) and separated with a permeable engraved line (15), wherein each compartment (14, 14’) can trap the same or a different liquid hydrogel precursor solution (12, 12’) liable to be in situ polymerized and comprises:

[0182] - said at least first hydrophilic spot (11) and said additional hydrophilic surface (Ila), and / or said hydrophobic spot (18); and

[0183] - optionally said second hydrophilic spot (11’),

[0184] and wherein each compartment (14’, 14’) optionally comprises an injection port (13, 13’).

[0185] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said chamber (8) being divided into two adjacent compartments (14, 14’), aligned with the axis made from opposite sides (8a, 8b) and separated with a permeable engraved line (15), wherein each compartment (14, 14’) can trap the same or a different liquid hydrogel precursor solution (12, 12’) liable to be in situ polymerized and comprises:

[0186] - said first and second hydrophilic spots (11, 11’); and

[0187] - said additional hydrophilic surface (Ila),

[0188] and wherein each compartment (14’, 14’) optionally comprises an injection port (13, 13’) (see Figure 8).

[0189] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein each compartment (14’, 14’) traps the same liquid hydrogel precursor solution (12, 12’) liable to be in situ polymerized.

[0190] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein each compartment (14’, 14’) traps a different liquid hydrogel precursor solution (12, 12’) liable to be in situ polymerized.

[0191] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein each compartment (14’, 14’) comprises an injection port (13, 13’).According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) comprises at least two independent chambers (8, 80), said chambers (8, 80):

[0192] - optionally comprising an injection port (13, 130);

[0193] - being adjacent to its own inlet (7, 70);

[0194] - being both in fluid communication with said outlet (5) \i.e. both said chambers (8, 80) are in fluid communication ,' and

[0195] - comprising at least said first hydrophilic spot (11, 110) and said additional hydrophilic surface (Ila, 110a), and / or said hydrophobic spot (18, 180), and optionally said second hydrophilic spot (IT, 110’),

[0196] wherein said chambers (8, 80) can trap the same or a different liquid hydrogel precursor solution (12, 120) liable to be in situ polymerized (see Figure 16).

[0197] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein each chamber (8, 80) traps the same liquid hydrogel precursor solution (12, 120) liable to be in situ polymerized.

[0198] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein each chamber (8, 80) traps a different liquid hydrogel precursor solution (12, 120) liable to be in situ polymerized.

[0199] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein each chamber (8, 80) comprises an injection port (13, 130).

[0200] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said microfluidic device (4) further comprises a baseless well-plate (16) stacked on the top layer (1), said baseless well-plate (16) comprising apertures (17) positioned above said inlet(s) (7, 9) and above said outlet (5) located in layers (1) and (2) (see Figure 6).

[0201] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said microfluidic device (4) further comprises a liquid hydrogel precursor solution (12) liable to be in situ polymerized.According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said microfluidic device (4) further comprises an in situ polymerized hydrogel (12a).

[0202] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said liquid hydrogel precursor solution (12) liable to be in situ polymerized or said in situ polymerized hydrogel (12a) comprise: prokaryotic cells, eukaryotic cells or a mixture thereof. Interestingly, when it is implemented two adjacent compartments (14, 14’) or at least two independent chambers (8, 80), as each compartment or each chamber may trap either the same liquid hydrogel precursor solution liable to be in situ polymerized or a different liquid hydrogel precursor solution liable to be in situ polymerized, each compartment or each chamber may thus trap either the same cells or different cells.

[0203] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said central well (6) is sealed using tape or cap or sticker.

[0204] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said central well (6) is sealed using transparent tape or transparent cap or transparent sticker. Advantageously, it has to be pointed out that transparency allows through the central well (6) the hydrogel chamber is imaged.

[0205] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein:

[0206] - said outlet (5) and said well (6) [= inlet (7) + chamber (8)] are filled with a medium, said medium diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8); or

[0207] - said outlet (5), said well (6) [= inlet (7) + chamber (8)] and optionally said channel (10) are filled with a medium, said medium diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8); or

[0208] - said well (6) [= inlet (7) + chamber (8)] is filled with a first medium, and said outlet (5), optionally said channel (10) and optionally said inlet (9) are filled with a second medium, said first and second media diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8),

[0209] in particular said medium comprising a compound, said compound being preferably a drug,in particular said first medium or said second medium comprising a compound, said compound being preferably a drug.

[0210] “Compound” refers to:

[0211] ■ potentially therapeutic molecules such as small molecules (e.g. antibiotics [e.g.

[0212] gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.),'

[0213] ■ peptides such as cytokines (e.g. TNF-a, IL-lb, IL-6, etc.);

[0214] ■ proteins such as antibodies (e.g. RituxanTM, HerceptinTM, HumiraTM, BenlystaTM, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.);

[0215] ■ etc.

[0216] “Drug” particularly refers to potentially therapeutic molecules such as small molecules (e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.).

[0217] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein:

[0218] - said outlet (5) and said well (6) are filled with a medium, said medium diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8); or - said outlet (5), said well (6) and optionally said channel (10) are filled with a medium, said medium diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8); or

[0219] in particular said medium comprising a compound, said compound being preferably a drug.

[0220] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said medium comprising a compound, said compound being preferably a drug. According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said medium comprising a drug.

[0221] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, wherein said well (6) is filled with a first medium, and said outlet (5), optionally said channel (10) and optionally said inlet (9) are filled with a second medium, said first and second media diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8),in particular said first medium or said second medium comprising a compound, said compound being preferably a drug.

[0222] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said first medium or said second medium comprising a compound, said compound being preferably a drug. According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said first medium or said second medium comprising a drug.

[0223] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said first medium comprising a compound, said compound being preferably a drug. According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said first medium comprising a drug.

[0224] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) being multiplexed. “Multiplexed” refers to the possibility of implement several chambers (8) and the means for operating them (outlet (5), inlets (7 optionally 9), optionally channel (10), etc.) on a single microfluidic device (4) of the invention (see Figures 5-8). In particular, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) being multiplexed and comprising 48 (see Figure 7A - single-well device) or 24 (see Figure 7B - two-well device) or 16 (see Figure 7C - three-well device) chambers (8) and the means for operating them.

[0225] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) being made of thermoplastic, in particular said thermoplastic being chosen among: poly(methyl methacrylate) (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), polypropylene (PP; also known as polypropene), polydimethylsiloxane (PDMS) and polyether ether ketone (PEEK).

[0226] According to another embodiment, the subject matter of the invention relates to the microfluidic device (4) as described above, said microfluidic device (4) being made of PMMA.

[0227] Interestingly, it has to be pointed out that the aforementioned use of at least a first and a second hydrophilic spots (11, IE) to implement a 3D pattern wetting so as to trap a liquid hydrogelprecursor solution (12) liable to be in situ polymerized within a microfluidic device (4) may be implemented with all the embodiments as described above.

[0228] According to a third aspect, the subject matter of the invention relates to a process of manufacturing a microfluidic device (4) as described above, said process comprising at least the following steps of:

[0229] a. manufacturing of three layers using laser cutting to obtain: a top layer (1), a microfluidic middle layer (2) and a bottom layer (3);

[0230] b. manufacturing a hydrophilic spot (11) to the top of said bottom layer (3) and optionally a hydrophilic spot (11’) to the bottom of said top layer (1) using:

[0231] ■ either a coating made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), polyethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof,

[0232] ■ or laser, or plasma, to obtained etched surfaces structured,

[0233] to obtain a top layer (1) optionally comprising a hydrophilic spot (IE) and a bottom layer (3) comprising a hydrophilic spot (11),

[0234] and / or

[0235] manufacturing a hydrophobic spot (18) to the top of said bottom layer (3) using:

[0236] ■ either a coating made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer,

[0237] ■ or a superhydrophobic surface, structured with micro / nano-texturization, to obtain a top layer (1) comprising a hydrophobic spot (18); and

[0238] c. assembling and bounding said top layer (1) optionally comprising a hydrophilic spot (11’), said microfluidic middle layer (2) and said bottom layer (3) comprising a hydrophilic spot (11) and / or a hydrophobic spot (18) to obtain said microfluidic device (4) as described above.

[0239] According to another embodiment, the subject matter of the invention relates to the process as described above of manufacturing a microfluidic device (4) as described above, said process comprising at least the following steps of:a. manufacturing of three layers using laser cutting to obtain: a top layer (1), a microfluidic middle layer (2) and a bottom layer (3);

[0240] b. manufacturing a hydrophilic spot (11) to the top of said bottom layer (3) and optionally a hydrophilic spot (11’) to the bottom of said top layer (1) using:

[0241] ■ either a coating made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), polyethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof,

[0242] ■ or laser, or plasma, to obtained etched surfaces structured,

[0243] to obtain a top layer (1) optionally comprising a hydrophilic spot (IE) and a bottom layer (3) comprising a hydrophilic spot (11); and

[0244] c. assembling and bounding said top layer (1) optionally comprising a hydrophilic spot (11’), said microfluidic middle layer (2) and said bottom layer (3) comprising a hydrophilic spot (11) to obtain said microfluidic device (4) as described above.

[0245] According to another embodiment, the subject matter of the invention relates to the process as described above of manufacturing a microfluidic device (4) as described above, said process comprising at least the following steps of:

[0246] a. manufacturing of three layers using laser cutting to obtain: a top layer (1), a microfluidic middle layer (2) and a bottom layer (3);

[0247] b. manufacturing a hydrophobic spot (18) to the top of said bottom layer (3) using:

[0248] ■ either a coating made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer,

[0249] ■ or a superhydrophobic surface, structured with micro / nano-texturization, to obtain a top layer (1) comprising a hydrophobic spot (18); and

[0250] c. assembling and bounding said top layer (1) optionally comprising a hydrophilic spot (11’), said microfluidic middle layer (2) and said bottom layer (3) comprising a hydrophobic spot (18) to obtain said microfluidic device (4) as described above.

[0251] According to another embodiment, the subject matter of the invention relates to the process as described above of manufacturing a microfluidic device (4) as described above, said process comprising at least the following steps of:a. manufacturing of three layers using laser cutting to obtain: a top layer (1), a microfluidic middle layer (2) and a bottom layer (3);

[0252] b. manufacturing a hydrophilic spot (11) to the top of said bottom layer (3) and optionally a hydrophilic spot (11’) to the bottom of said top layer (1) using:

[0253] ■ either a coating made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), polyethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof,

[0254] ■ or laser, or plasma, to obtained etched surfaces structured,

[0255] to obtain a top layer (1) optionally comprising a hydrophilic spot (IE) and a bottom layer (3) comprising a hydrophilic spot (11),

[0256] and

[0257] manufacturing a hydrophobic spot (18) to the top of said bottom layer (3) using:

[0258] ■ either a coating made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer,

[0259] ■ or a superhydrophobic surface, structured with micro / nano-texturization, to obtain a top layer (1) comprising a hydrophobic spot (18); and

[0260] c. assembling and bounding said top layer (1) optionally comprising a hydrophilic spot (11’), said microfluidic middle layer (2) and said bottom layer (3) comprising a hydrophilic spot (11) and a hydrophobic spot (18) to obtain said microfluidic device (4) as described above.

[0261] According to another embodiment, the subject matter of the invention relates to the process as described above of manufacturing a microfluidic device (4) as described above, said process comprising at least the following steps of:

[0262] a. manufacturing of three layers using laser cutting to obtain: a top layer (1), a microfluidic middle layer (2) and a bottom layer (3);

[0263] b. manufacturing a hydrophilic spot (11) to the top of said bottom layer (3) and a hydrophilic spot (11’) to the bottom of said top layer (1) using:

[0264] ■ either a coating made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), polyethylene glycol) methacrylate (PEGMA),poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof,

[0265] ■ or laser, or plasma, to obtained etched surfaces structured (hydrophilic rough surfaces),

[0266] to obtain a top layer (1) comprising a hydrophilic spot (11) and a bottom layer (3) comprising a hydrophilic spot (11’); and

[0267] c. assembling and bounding said top layer (1) comprising a hydrophilic spot (11), said microfluidic middle layer (2) and said bottom layer (3) comprising a hydrophilic spot (11’) to obtain said microfluidic device (4) as described above.

[0268] According to another embodiment, the subject matter of the invention relates to the process as described above of manufacturing a microfluidic device (4) as described above, wherein step b. further comprises manufacturing an additional hydrophilic surface (Ila) to the top of said bottom layer (3) using:

[0269] ■ either a coating made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), polyethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof,

[0270] ■ or laser, or plasma, to obtained etched surfaces structured,

[0271] to obtain a bottom layer (3) further comprising an additional hydrophilic surface (Ila), in particular said additional hydrophilic surface (Ila) being contiguous with said hydrophilic spot (H).

[0272] According to another embodiment, the subject matter of the invention relates to the process as described above of manufacturing a microfluidic device (4) as described above, wherein step b. further comprises manufacturing a third hydrophilic spot (101) placed at the surface of said side (8c) and a fourth hydrophilic spot (1001) placed at the surface of said side (8d) using:

[0273] ■ either a coating made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), polyethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof,

[0274] ■ or laser, or plasma, to obtained etched surfaces structured,to obtain a bottom layer (3) further comprising an additional hydrophilic surface (Ila), in particular said additional hydrophilic surface (Ila) being contiguous with said hydrophilic spot (H).

[0275] According to another embodiment, the subject matter of the invention relates to the process of manufacturing as described above, said process further comprising a step of injection of a liquid hydrogel precursor solution (12) into a chamber (8) through an inlet (7), said liquid hydrogel precursor solution (12) polymerizing in situ, to obtain a microfluidic device (4) as described above,

[0276] in particular said liquid hydrogel precursor solution (12) comprising: prokaryotic cells, eukaryotic cells or a mixture thereof.

[0277] Interestingly, the step of injection of a liquid hydrogel precursor solution (12) into a chamber (8) is a manual injection or is a robotized injection by using an automated droplet printer or dispenser. Consequently, another embodiment of the invention relates to the process of manufacturing as described above, wherein said step of injection is a manual injection or is a robotized injection by using an automated droplet printer or dispenser. In particular, a subject matter of the invention relates to the process of manufacturing as described above, wherein said step of injection is a robotized injection by using an automated droplet printer or dispenser.

[0278] According to another embodiment, the subject matter of the invention relates to the process of manufacturing as described above, wherein said liquid hydrogel precursor solution (12) comprises: prokaryotic cells, eukaryotic cells or a mixture thereof. “Mixture thereof’ refers to a mixture of prokaryotic- and eukaryotic- cells.

[0279] “Prokaryotic cells” refers to cells that exhibit characteristics of prokaryotes, which is a term well known in the art. Prokaryotes are typically unicellular organisms and lack organelles (such as mitochondria, chloroplasts, and Golgi apparatus), a cytoskeleton, and a discrete nucleus. Examples of prokaryotic cells include bacteria, such as eubacteria, cyanobacteria and prochiorophytes; archaebacteria; and other microorganisms such as rickettsias, mycoplasmas, spiroplasmas, and chlamydiae. Preferably, the prokaryotic cells are bacteria (E. coli. P. aeruginosa, etc.). According to another embodiment, the subject matter of the invention relates to the process of manufacturing as described above, wherein said liquid hydrogel precursor solution (12) comprises prokaryotic cells.“Eukaryotic cells” refers to refers to cells that exhibit characteristics of eukaryotes, which is a term well known in the art. Examples of eukaryotic cells are mammalian cells (in particular human cells), insect cells, plant cells, yeast cells and fungal cells. According to another embodiment, the subject matter of the invention relates to the process of manufacturing as described above, wherein said liquid hydrogel precursor solution (12) comprises eukaryotic cells. In particular, the subject matter of the invention relates to the process of manufacturing as described above, wherein said liquid hydrogel precursor solution (12) comprises human cells (derived from primary culture or cell line).

[0280] To illustrate the design and fabrication of the microfluidic device (4) of the invention, the following details are provided:

[0281] 1.1 Design

[0282] The device has been designed using Clewin 5 software. The schematic illustration of a 48 well plate consisting of a 4x6 arrayed 2-well device is shown in Figure 5. The device design can be scaled up for different well plates, and dimensions will be changed according to the available well area. The microfluidic device (4) consists of three layers (1, 2 and 3) assembled / bonded upon one another and then to the base of the baseless well-plate (16) (Figure 6A). The layer materials are thermoplastics and are bonded using 3M epoxy® tape. Examples of thermoplastics are PMMA, PS, PC, COC, PP, etc. The bottom layer (3) is the base and is used for liquid guiding. This bottom layer (3) (plane 3') has a hydrophilic spot (11) that acts as a base of a hydrogel chamber (8). The chamber base is trapezoidal, and its dimensions are 1.5 mm x 0.5 mm, 1.5 mm (parallel side 1, parallel side 2, distance between parallel sides respectively). Additionally, the bottom layer (3) has an extended engraved section (Ila = extra edge) (1.5 mm x 0.1 - 0.25 mm; length, width respectively), as shown in Figure 4 and 6B, which is open to the well plate top and allows the printing and suction of hydrogel when operated in automated mode. The microfluidic middle layer (2) is the experimental layer, which holds the hydrogel (12a) incorporated with cells. The microfluidic middle layer (2) area dimension is the same as that of the hydrogel chamber (8) and has a height of 0.25 mm. The microfluidic middle layer (2) is connected to microfluidics channels (10) (width - 0.2 mm) to guide the media / drugs in and out of the microfluidic device (4). One of the faces of the hydrogel chamber (8) acts allows drug diffusion, as shown in Figure 6C. The drug injection port (7 / 13) allows the antibiotics and fresh media to be in contact with the hydrogel (12a). The media / antibiotics pass across the hydrogel (12a) during the flow and are collected at the outlet (5) well of the device for further analysis. In perfusion-configured devices, the fresh media, once it crosses the hydrogel (12a),never goes back to the hydrogel (12a). The top layer (1) has the inlet (7) and outlet (5) ports and is additionally used as a liquid guide (hydrophilic spot). The inlet (7) ports are for media / antibiotics injection, and the outlet (5) port is used for media and antibiotics collection. The top layer (1) has a hemispherical hydrogel injection port (13) that facilitates the injection process (Figure 6D). Moreover, this top layer (1) (plane 1) has a similar hydrophilic spot (11’) of the same dimension as that of the bottom layer (3). Here the hydrophilic spot (11’) acts as a top section of hydrogel (12a). In this way, both the hydrophilic spot, base (11), and top (11’) allow the trapping of hydrogel (12a) and holding intact in the chamber area (8). Further, the top layer (1) (plane 1') is bonded to the base of the baseless well-plate (16). This allows the microfluidic device (4) to hold the media / antibiotics in the wells and create a hydrostatic flow. The placement of the chamber (8) in the design is done keeping in mind the ease of injection and imagining. The chamber (8) is placed off-centered, as shown in Figure 6A. This is done to make sure while users are doing the manual injection, the entire tip of the pipette is within the vicinity of the hydrophilic spot (11, 11’). This is done so that all the hydrogel (12a) gets trapped within the chamber (8) and wastage is minimal. The imagining of the cells in a hydrogel (12a) is additionally a crucial factor that drives the placement of the chamber (8). The shadow of the well, while imagining, shouldn’t interfere so that the chamber (8) is not placed at the periphery of the base well and is kept in between the center and periphery.

[0283] 1.2 Fabrication using Laser cutter

[0284] The design is made using Clewin software, and layers are created by cutting using a CO2 laser (AxysLaser). The 2-well design for each layer is shown in Figure 6. The innovation uses thermoplastic PMMA (thickness- 0.25 mm) to create the microfluidic device (4). The fabrication can be extended to other thermoplastics like polystyrene, PEEK, etc. The hydrophilic spot area (11, 11’, Ila), as shown in Figure 6B, D in layers (1) and (3) (plane (1) and 3’ respectively), is just engraved using low power (power 8W and speed 5.08 cm / sec) to create a guiding liquid interface. The interface of hydrogel (12a) remains attached to these engraved lines. All other designs that required the laser to cut through are done at power 28W and speed 3.04 cm / sec. The microfluidic middle layer (2), as shown in Figure 6A, C, has an epoxy layer at both the faces, 2 and 2'. The microfluidic middle layer (2) with epoxy is then used for laser cutting (power - 36W; speed - 2.03cm / sec) to create the design. The airflow mode is kept switched on to remove the plastic residue while cutting. After cutting, the layers are cleaned thoroughly with 50% ethanol water solution, followed by nitrogen drying. The PMMA layer’s thickness will be the hydrogel chamber’s desired height.1.3 Coating to make the hydrophilic spot and / or the hydrophobic spot

[0285] The engraved spots (1, 3’) in layers (1) and (3) are coated using hydrophilic liquid. The hydrophilic spot (11, 11’, Ila) can be created by coating biocompatible hydrophilic liquids (DMEM, MEM, RPMI, F-12, OPTI-MEM, MCCOY’S 5 A MEDIUM, KSFM, etc.) or laser engraving. The coating can be done using manual or automatic techniques. In the manual coating process, the desired amount of liquid (0.5 pL) is placed using the pipette. The design layer with coated liquid is then placed in a hotplate at 50°C to dry the liquid. The process is done twice to have a uniform layer. In the automated technique, a mask of the size of chamber size is placed above layers (1) and (3). A laser with high power and microsecond emission time is used to form a hydrophilic spot (11, 11’, Ila) in the chamber (8).

[0286] The hydrophobic spot (18) is prepared on the top of the layer (3) at the location designated to form the lower surface of the inlet (7) by selective deposition or surface treatment. Techniques include (but are not limited to): vapor-phase deposition of perfluoroalkylsilanes (silanization), plasma deposition or plasma fluorination, deposition of a fluoropolymer by spin / spray or vapor coating (e.g., Teflon™-like films), or laser ablation to generate micro / nano roughness followed by application of a hydrophobic chemical. Where selective treatment is desired, the chamber (8) interior may be masked (for example with micro-masking tape or a removable mask) prior to application of the hydrophobic treatment so that the chamber (8) interior remains untreated or is separately coated with a hydrophilic material (11, 11’). After hydrophobic treatment, the masked areas are removed, the device layers cleaned (50% ethanol followed by nitrogen drying as described elsewhere) and assembled as described.

[0287] 1.4 Assembly and Bonding

[0288] The layers (1, 2, 3), upon coating, are now aligned using an aligned frame and microscope. The aligned layers (1, 2, 3) were bonded by removing the sacrificial layer from the epoxy tape. The bonded layers (1, 2, 3) are subjected to uniform pressure to remove the trapped air and higher bonding strength. The microfluidic device (4) thus formed is bonded to a baseless well-plate (16) using epoxy tape. The baseless well-plate (16) is connected directly to the device port for operation.

[0289] According to another aspect, the subject matter of the invention relates to the use of a hydrophilicity difference to implement a 3D pattern wetting so as to trap a liquid hydrogelprecursor solution (12) liable to be in situ polymerized within a microfluidic device (4) as described above.

[0290] Alternatively, this aspect concerns the use a microfluidic device (4) as described above to implement a 3D pattern wetting so as to trap a liquid hydrogel precursor solution (12) liable to be in situ polymerized, said 3D pattern wetting being a hydrophilicity difference between the surface of said chamber (8) and the surface of said at least one inlet (7), said hydrophilicity being measured by contact angle method.

[0291] According to another embodiment, the subject matter of the invention relates to the use of a hydrophilicity difference as described above, wherein:

[0292] - at least a first hydrophilic spot (11) is placed at the lower surface of said chamber (8) and extends contiguously towards the lower surface of said inlet (7) so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0293] said first hydrophilic spot (11):

[0294] ■ being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2- hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0295] ■ being etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8);

[0296] and / or

[0297] - a hydrophobic spot (18) is placed at the lower surface of said inlet (7) so as to promote the confinement of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),

[0298] said hydrophobic spot (18):

[0299] ■ being made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; or

[0300] ■ being a superhydrophobic surface, structured with micro / nano- texturization on the lower surface of said inlet (7).According to another embodiment, the subject matter of the invention relates to the use of a hydrophilicity difference as described above, wherein a second hydrophilic spot (11’) is placed at the top surface of said chamber (8),

[0301] said second hydrophilic spot (IT):

[0302] - being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0303] - being etched surfaces structured with a laser or a plasma on the top surface of said chamber (8).

[0304] According to another embodiment, the subject matter of the invention relates to the use of a hydrophilicity difference as described above, wherein a third hydrophilic spot (101) is placed at the surface of said side (8c) and a fourth hydrophilic spot (1001) is placed at the surface of said side (8d),

[0305] said third hydrophilic spot (101) and said fourth hydrophilic spot (1001):

[0306] - being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or

[0307] - being etched surfaces structured with a laser or a plasma on the surfaces of said sides (8c, 8d) (see Figure 17).

[0308] According to another aspect, the subject matter of the invention relates to the use of a microfluidic device (4) as described above for in vitro cell culture and / or compound screening, in particular said cell being chosen among: prokaryotic cells, eukaryotic cells and a mixture thereof,

[0309] in particular said compound being a drug.

[0310] According to another embodiment, the subject matter of the invention relates to the use of a microfluidic device (4) as described above for in vitro cell culture, said cell being chosenamong: prokaryotic cells, eukaryotic cells and a mixture thereof. In particular, the subject matter of the invention relates to the use of a microfluidic device (4) as described above for in vitro cell culture, said cell being eukaryotic cells.

[0311] According to another embodiment, the subject matter of the invention relates to the use of a microfluidic device (4) as described above for compound screening. In particular, the subject matter of the invention relates to the use of a microfluidic device (4) as described above for compound screening, said compound being a drug.

[0312] According to another embodiment, the subject matter of the invention relates to the use of a microfluidic device (4) as described above for in vitro cell culture and compound screening, said cell being chosen among: prokaryotic cells, eukaryotic cells and a mixture thereof. In particular, the subject matter of the invention relates to the use of a microfluidic device (4) as described above for in vitro cell culture and compound screening, said cell being eukaryotic cells. In particular, the subject matter of the invention also relates to the use of a microfluidic device (4) as described above for in vitro cell culture and compound screening, said compound being a drug.

[0313] According to another embodiment, the subject matter of the invention relates to the use as described above, said in vitro cell culture being a coculture [which involve a microfluidic device (4) as illustrated, e.g., Figure 8],

[0314] According to another aspect, the subject matter of the invention relates to a method for generating one diffusive compound gradient in a chamber (8) located in a microfluidic device (4) as described above, said method comprising at least the following steps of:

[0315] a. filling a well (6) [= inlet (7) + chamber (8)] with a first medium comprising said compound, said first media comprising said compound diffusing in an in situ polymerized hydrogel (12a) located in said chamber (8) through a side (8a); and b. filling an inlet (9), which is in fluid connection with an outlet (5) and a channel (10), with a second medium lacking said compound, said second medium lacking said compound diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8) through a side (8b),

[0316] in particular said compound being a drug.Alternatively, this aspect concerns the use a microfluidic device (4) as described above for generating one diffusive compound gradient in a chamber (8), said use comprising at least the following steps of:

[0317] a. filling a well (6) with a first medium comprising said compound, said first media comprising said compound diffusing in an in situ polymerized hydrogel (12a) located in said chamber (8) through a side (8a); and

[0318] b. filling an inlet (9), which is in fluid connection with an outlet (5) and a channel (10), with a second medium lacking said compound, said second medium lacking said compound diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8) through a side (8b),

[0319] in particular said compound being a drug.

[0320] It has to be pointed out that such method involves the use of a microfluidic device (4) in the three-well configuration (see Figures 3-4, 7C and 9) since an inlet (9) and a channel (10) are needed (see Example 3.3.2).

[0321] “Diffusive compound gradient” refers to the fact that the circulation of liquids in the three-well microfluidic device (4) of the invention, especially those containing and carrying said compound, generates a gradient of concentration of said compound in the chamber (8). “Gradient of concentration” refers to the concentration of said compound from one point to another of said chamber (8) varies.

[0322] According to another embodiment, the subject matter of the invention relates to a method for generating one diffusive compound gradient as described above, wherein said compound is a drug.

[0323] In any event, it should be noted that the various aspects of the invention, as well as the various embodiments thereof, are interdependent. These can therefore be combined with each other to obtain preferred aspects and / or embodiments of the invention not explicitly described. This is also true for the set of definitions provided in this description, which applies to all aspects of the invention and its embodiments.

[0324] Furthermore, the present invention is illustrated by, but not limited to, the following Figures and Examples.LIST OF FIGURES

[0325] Figure 1. Schematic of one-well device configuration.

[0326] Figure 2. Schematic of two-well device configuration.

[0327] Figure 3. Schematic of three-well device configuration. Inset shows the formation of a gradient in the three-well device when the transparent cap is used in the central well.

[0328] Figure 4. Schematic of three-well device configuration.

[0329] Figure 5. Two-well device in a 48 well plate configuration with inset showing the single device and magnified version of hydrogel chamber.

[0330] Figure 6. Schematic of two-well device. (A) Different layers used to build the device with baseless well-plate at the top. (B) layer 3 (C) layer 2 (D) layer 1.

[0331] Figure 7. Schematic of different device configurations (A) one-well device; (B) two-well device; (C) three-well device; Inset shows the formation of a gradient in the three-well device when the transparent cap is used in the central well.

[0332] Figure 8. Co-culture two-well device design in 48 well plate configuration. There are two different hydrogel injection points where different cell types can be seeded.

[0333] Figure 9. Schematic of the two-well device showing the flow mechanism. (A) hydrostatic flow for two-well devices and three-well devices. (B) single-dose drug screening. (C) gradient formation in the three-well device.

[0334] Figure 10. Schematic of Operating protocol. (A) different injection processes of seeding hydrogel, media, and drugs; (B) the injection technique; (C) the cell recovery process.

[0335] Figure 11. Schematic of drop printer interface with two-well device. Show the injection and hydrogel trapping when a droplet from the printer is placed in the extended hydrophilic section.Figure 12. 3D Cell morphology results.

[0336] Figure 13. Schematic of coating design configuration.

[0337] Figure 14. Schematic of chamber design configuration.

[0338] Figure 15. Schematic of two-well device encompassing a cured porous polymer network layer (50) located in outlet (5).

[0339] Figure 16. Co-culture two-well device configuration with two chambers (8, 80).

[0340] Figure 17. Schematic of coating design configuration.

[0341] Common legend for figures 1-10:

[0342] 1. Top layer

[0343] 2. Microfluidic middle layer

[0344] 3. Bottom layer

[0345] 4. Microfluidic device

[0346] 5. Outlet or media / drug outlet

[0347] 6. Well or central well

[0348] 7. Inlet or drug inlet or drug injection port

[0349] 8. 80. Chamber or hydrophilic hydrogel chamber or liquid / hydrogel trapping zone

[0350] 9. Inlet or media inlet

[0351] 10. Channel

[0352] 11, 11’, 110. Hydrophilic spot

[0353] Ila, 110a. Additional hydrophilic surface or extra edge or extended engraved section

[0354] 12, 12’, 120. Liquid hydrogel precursor solution

[0355] 12a, 12a’. Hydrogel or in situ polymerised hydrogel

[0356] 13, 13’, 130. Injection port

[0357] 14, 14’. Compartment

[0358] 15. Engraved line

[0359] 16. Baseless well-plate17. Aperture

[0360] 18. Hydrophobic spot

[0361] EXAMPLES

[0362] 1. Design and fabrication of device

[0363] 1.1 Design

[0364] The device has been designed using Clewin 5 software. The schematic illustration of a 48 well plate consisting of a 4x6 arrayed 2-well device is shown in Figure 5. The device design can be scaled up for different well plates, and dimensions will be changed according to the available well area. The microfluidic device (4) consists of three layers (1, 2 and 3) assembled / bonded upon one another and then to the base of the baseless well-plate (16) (Figure 6A). The layer materials are thermoplastics and are bonded using 3M epoxy® tape. Examples of thermoplastics are PMMA, PS, PC, COC, PP, etc. The bottom layer (3) is the base and is used for liquid guiding. This bottom layer (3) (plane 3') has a hydrophilic spot (11) that acts as a base of a hydrogel chamber (8). The chamber base is trapezoidal, and its dimensions are 1.5 mm x 0.5 mm, 1.5 mm (parallel side 1, parallel side 2, distance between parallel sides respectively). Additionally, the bottom layer (3) has an extended engraved section (1 la = extra edge) (1.5 mm x 0.1 - 0.25 mm; length, width respectively), as shown in Figure 4 and 6B, which is open to the well plate top and allows the printing and suction of hydrogel (12a) when operated in automated mode. The microfluidic middle layer (2) is the experimental layer, which holds the hydrogel (12a) incorporated with cells. The microfluidic middle layer (2) area dimension is the same as that of the hydrogel chamber (8) and has a height of 0.25 mm. The microfluidic middle layer (2) is connected to microfluidics channels (10) (width - 0.2 mm) to guide the media / drugs in and out of the microfluidic device (4). One of the faces of the hydrogel chamber (8) acts allows drug diffusion, as shown in Figure 6C. The drug injection port (7 / 13) allows the antibiotics and fresh media to be in contact with the hydrogel (12a). The media / antibiotics pass across the hydrogel (12a) during the flow and are collected at the outlet (5) well of the device for further analysis. In perfusion-configured devices, the fresh media, once it crosses the hydrogel (12a), never goes back to the hydrogel (12a). The top layer (1) has the inlet (7) and outlet (5) ports and is additionally used as a liquid guide (hydrophilic spot). The inlet (7) ports are for media / antibiotics injection, and the outlet (5) port is used for media and antibioticscollection. The top layer (1) has a hemispherical hydrogel injection port (13) that facilitates the injection process (Figure 6D). Moreover, this top layer (1) (plane 1) has a similar hydrophilic spot (11’) of the same dimension as that of the bottom layer (3). Here the hydrophilic spot (11’) acts as a top section of hydrogel (12a). In this way, both the hydrophilic spot, base (11), and top (11’) allow the trapping of hydrogel (12a) and holding intact in the chamber area (8). Further, the top layer (1) (plane 1') is bonded to the base of the baseless well-plate (16). This allows the device to hold the media / antibiotics in the wells and create a hydrostatic flow. The placement of the chamber (8) in the design is done keeping in mind the ease of injection and imagining. The chamber (8) is placed off-centered, as shown in Figure 6A. This is done to make sure while users are doing the manual injection, the entire tip of the pipette is within the vicinity of the hydrophilic spot (11, 11’). This is done so that all the hydrogel (12a) gets trapped within the chamber (8) and wastage is minimal. The imagining of the cells in a hydrogel is additionally a crucial factor that drives the placement of the chamber (8). The shadow of the well, while imagining, shouldn’t interfere so that the chamber (8) is not placed at the periphery of the base well and is kept in between the center and periphery.

[0365] 1.2 Fabrication using Laser cutter

[0366] The design is made using Clewin software, and layers are created by cutting using a CO2 laser (AxysLaser). The 2-well design for each layer is shown in Figure 6. The innovation uses thermoplastic PMMA (thickness- 0.25 mm) to create the microfluidic device (4). The fabrication can be extended to other thermoplastics like polystyrene, PEEK, etc. The hydrophilic spot area (11, 11’, Ila), as shown in Figure 6B, D in layers (1) and (3) (plane (1) and 3’ respectively), is just engraved using low power (power 8W and speed 5.08 cm / sec) to create a guiding liquid interface. The interface of hydrogel (12a) remains attached to these engraved lines. All other designs that required the laser to cut through are done at power 28W and speed 3.04 cm / sec. The microfluidic middle layer (2), as shown in Figure 6A, C, has an epoxy layer at both the faces, 2 and 2'. The microfluidic middle layer (2) with epoxy is then used for laser cutting (power - 36W; speed - 2.03cm / sec) to create the design. The airflow mode is kept switched on to remove the plastic residue while cutting. After cutting, the layers are cleaned thoroughly with 50% ethanol water solution, followed by nitrogen drying. The PMMA layer’s thickness will be the hydrogel chamber’s desired height.

[0367] 1.3 Coating to make the hydrophilic spot and / or the hydrophobic spotThe engraved spots (1, 3’) in layers (1) and (3) are coated using hydrophilic liquid. The hydrophilic spot (11, 11’, Ila) can be created by coating biocompatible hydrophilic liquids (DMEM, MEM, RPMI, F-12, OPTI-MEM, MCCOY’S 5 A MEDIUM, KSFM, etc.) or laser engraving. The coating can be done using manual or automatic techniques. In the manual coating process, the desired amount of liquid (0.5 pL) is placed using the pipette. The design layer with coated liquid is then placed in a hotplate at 50°C to dry the liquid. The process is done twice to have a uniform layer. In the automated technique, a mask of the size of chamber size is placed above layers (1) and (3). A laser with high power and microsecond emission time is used to form a hydrophilic spot (11, 11’, Ila) in the chamber (8).

[0368] The hydrophobic spot (18) was prepared on the top of the layer (3) at the location designated to form the lower surface of the inlet (7) by selective deposition or surface treatment. Techniques include (but are not limited to): vapor-phase deposition of perfluoroalkylsilanes (silanization), plasma deposition or plasma fluorination, deposition of a fluoropolymer by spin / spray or vapor coating (e.g., Teflon™-like films), or laser ablation to generate micro / nano roughness followed by application of a hydrophobic chemical. Where selective treatment was desired, the chamber (8) interior may be masked (for example with micro-masking tape or a removable mask) prior to application of the hydrophobic treatment so that the chamber (8) interior remains untreated or is separately coated with a hydrophilic material (11, 11’). After hydrophobic treatment, the masked areas were removed, the device layers cleaned (50% ethanol followed by nitrogen drying as described elsewhere) and assembled as described.

[0369] 1.4 Assembly and Bonding

[0370] The layers (1, 2, 3), upon coating, are now aligned using an aligned frame and microscope. The aligned layers (1, 2, 3) were bonded by removing the sacrificial layer from the epoxy tape. The bonded layers (1, 2, 3) are subjected to uniform pressure to remove the trapped air and higher bonding strength. The microfluidic device (4) thus formed is bonded to a baseless well-plate (16) using epoxy tape. The baseless well-plate (16) is connected directly to the device port for operation.

[0371] 1.5 Different design configuration

[0372] The innovation has different design configurations. The design can be categorized based on perfusion types and drug dose systems. The innovation shows that the design can be adopted both in perfusion and non-perfusion-based. The perfusion-less device shown in Figures 1 and7A consists of a single-well where the media / antibiotic solution is stationary, and the flow doesn’t happen. This configuration will have throughput to that of the well plate number, and the drug combination is limited to a single dose per well. For perfusion devices shown in Figures 2, 3 and 7B, C, there can be two- or three-wells’ designs. In two-well devices, the flow occurs from the drug port (7) to the outlet (5). The throughput here is half of the well plate number. Like a single-well device, a 2-well device can be used for a single-dose drug study. The only difference between single and two-well designs is the flow (perfusion). The three-well device has a separate well for drug injection (7) and separate wells for media inlet (9) and outlet (5) (Figures 3, 4 and 7C). Here, the microfluidic device (4) can form a drug gradient when the media inlet (9) and outlet (5) flow are optimized. The drug port (7) contains the antibiotic solution and is stationary. The flow of antibiotics across the hydrogel (12a) occurs purely by diffusion, and media flow takes out the antibiotics from the microfluidic device (4), creating a stable gradient throughout the experiment. In this configuration, the central drug well (6) is sealed using transparent tape after the drug injection process. This is done so that the flow of media should be established between the inlet well (9) and the outlet well (5) and keep the central well (6) stationary. The throughput of this design is one-third of the well plate number.

[0373] 2. Establishing perfusion: Hydrostatic and diffusive flow

[0374] There are two types of flow in the innovation. The first is hydrostatic pressure-based (Figure 9 A), and the second is purely diffusion-based (Figure 9A, C). The hydrostatic pressure flow is generated using the height of the liquid in the wells. The height creates a pressure difference between the inlet (9) and outlet (5), and flow takes place. The flow across the hydrogel (12a), which is porous in nature, takes place due to this pressure difference and resistance by the hydrogel (12a) itself. This kind of flow is mainly in one-well and two-well devices, and media flow is in three-well devices. The estimated time to balance out the inlet (9) and outlet (5) wells is 18 hours. The purely diffusive flow is mainly in three-well plate devices where the drug port (7) liquid is stationary, and flow across the hydrogel (12a) occurs due to the diffusion of antibiotics.

[0375] 3. Operating Protocol3.1 Injection of hydrogel

[0376] The injection of hydrogel uses the principle of capillary trapping.

[0377] The hydrogel (12a) injection can be done in two ways, as shown in Figure 10. The first is manual injection. In this process, a desired volume (0.5pL) of liquid hydrogel precursor solution (12) (depending on chamber geometry) is placed close to the hydrophilic spot (11, 11’, Ila) using a pipette. A little pressure is exerted to take the liquid hydrogel precursor solution (12) out of the tip, and the wettability of the chamber (8) sucks the liquid hydrogel precursor solution (12) inside (Figure 10). The pipette tip is tilted to place the tip close to the hydrophilic spot (11, 11’, Ila) using the tip injection point (Figure 10B). The second way is by using an automated droplet printer or dispenser. In this, a droplet of the desired volume is placed just at the extended section of the hydrophilic spot (11, 11’, Ila), as shown in Figure 11. The hydrophilicity of the chamber (8) sucks the liquid hydrogel precursor solution (12) inside the channel. In this way, hydrogel injection is automated using a droplet printer or dispenser. The top layer hydrophilic spot (11’) is designed to facilitate the droplet to fall close to the hydrophilic area, as shown in Figure 11. Once the liquid hydrogel precursor solution (12) is injected, the device is kept at an elevated temperature of 37°C for 5 minutes inside an incubator to cure in situ the hydrogel (12a).

[0378] Operation with hydrophobic surroundings. When the the lower surface of said inlet (7) comprises a hydrophobic spot (18), injection may rely on the hydrophilicity difference (= wettability contrast): droplets deposited near the chamber (8) inlet should spontaneously remain confined to the chamber (8) interior due to the hydrophobic perimeter. In practice, the printing / droplet-deposition step is identical, but droplets that contact the hydrophobic spot (18) will not spread outward.

[0379] 3.2 Media perfusion

[0380] The media is poured into the wells once the hydrogel (12a) (containing cells) is cured inside the chamber (8). The media flow across the hydrogel (12a), allowing cells to thrive in a new environment. This process takes place for 36 hours. The media is replaced from the outlet (5) well after every 12 hours, thus keeping the flow throughout the experiment.3.3 Drug Injection

[0381] To perform the drug study, the drug solution has to be introduced into the microfluidic device (4). For this, two different types of studies can be performed which is based on device design:

[0382] 3.3.1 Single dose

[0383] First, the media is taken out, and fresh media with dissolved drugs is introduced in the inlet port (7). A single-dose study means a single concentration of drug study on a single device. The diffusion of drugs across the hydrogel (12a) interface leads to uniform drug distribution throughout the chamber after reaching equilibrium (Figure 9B). Single-well and two-well devices are used for this kind of study. The drug study usually takes place for 4 days, and the media / drug solutions from the outlet (5) are taken out every 12 hours.

[0384] 3.3.2 Gradient formation

[0385] Three-well devices are used to study the drug gradient. After removing the media from the microfluidic device (4), a drug solution is filled at the center well (6), allowing the drug to diffuse into the hydrogel (12a) (Figure 10A). The central well (6) is sealed using a transparent cap (Figure 9A) or sticker. Following this, the media well (inlet well (9)) is filled with culture media. This can be done manually using a pipette or automatically with a robotic liquid dispenser. Sealing the middle layer ensures a controlled flow between the first and third wells, allowing media to pass over the hydrogel (12a) interface. This setup promotes diffusion through the hydrogel (12a) without any convective flow. The media with the drug in the central well (6) acts as the source, and as it flows through the system, the media in the sink removes the drug with the flow. This setup maintains a constant diffusive gradient across the hydrogel (12a) (Figure 9C).

[0386] 3.3.2 Recovering the cells

[0387] After the drug study, the cells in this innovation can be recovered (two-well device) by applying an air jet from the outlet well (5) and recovering the cells from the inlet port (7) (Figure 10C).

[0388] This way, the air jet breaks the hydrogel (12a), and part comes out in the inlet chamber (7).4. Interfacing with Droplet Printer

[0389] The interfacing with droplet printers is a crucial aspect of this innovation. The extended section (Ila) placed on the base layer allows the droplet trapping. The extended engraved section of dimensions (1.5 mm x 0.25 mm) is placed in such a way that the dispensing droplet, when in contact with the drug inlet port (7) (in the case of a two-well plate), the droplet’s one-fourth periphery area touches the hydrophilic section (Figure 11). This placement mechanism led the droplet to come in contact with the hydrophilic section (11, 11’, Ila) and allowed the suction of the remaining droplet volume (capillary trapping) inside the chamber (8). This process allows the individual droplets to print inside confined cavities (Figure 11). Using this configuration, all other wells can be automatically filled using droplet printers or dispensers.

[0390] 5. Cell in 3D hydrogel chamber

[0391] For a 48-well plate design, the injected volume is 5 pL for chamber dimensions 1.5 mm x 0.5 mm, 1.5 mm x 0.25 mm (length 1, length 2, width, height respectively). Cell density also varies depending on the cell type and chamber volume. For instance, with the A549 cell line, a typical number of 1,000 cells is necessary when injecting 5 pL of liquid hydrogel precursor solution (12). The concentration of the hydrogel (12a) is critical, as an optimal concentration allows cells to migrate freely within it. When using Matrigel, we dilute it to 50%. Hydrogel concentration can be adjusted based on the cell type and number. A 50% diluted Matrigel cures in about 5 minutes at 37°C. The cells inside the matrigel at different z position is shown Figure 12A and the formation of spheroids and cell network after 15 days of culturing is shown in Figure 12B.

[0392] 6. Coculture Design and operation

[0393] This innovation has a co-culture design that can culture two different cell types. The design has two small changes, as shown in Figure 8. The base (3) and top (1) layer have an engraved straight line (15) mark at the center of the trapezium, and the second one is that the device has two hemispherical injection points (13, 13’) in the top layer (1). The trapping mechanism is the same. First, a liquid hydrogel precursor solution (12) containing cell type A is injected from one port (13), and then liquid hydrogel precursor solution (12’) with cell type B is injected fromthe second injection point (13’). After the first injection, the center engraved straight line (15) in the trapezium chamber (8) will hold the first hydrogel (12a), giving space for the second hydrogel (12a’) to trap. Everything else remains the same in this design.

Claims

CLAIMS1. Microfluidic device (4) made up of a stack of at least three layers (1, 2, 3):a top layer (1) containing hollow spaces;a microfluidic middle layer (2) containing hollow spaces; anda bottom layer (3)the stacking of these three layers (1, 2, 3) defining at least one inlet (7) adjacent to a chamber (8), said chamber (8) forming a trapping zone for a liquid hydrogel precursor solution (12) liable to be in situ polymerized,said chamber (8) comprising:- a lower face;- a top face; and- 4 sides (8a, 8b, 8c, 8d), the side (8a) being opposed to the side (8b) and the side (8c) being opposed to the side (8d),said chamber (8) being:- opened on 2 opposite sides (8a, 8b), the side (8a) being adjacent to said at least one inlet (7);- in direct fluid communication with said at least one inlet (7) through said side (8a); and- located in layer (2) and between layers (1) and (3),wherein at least the surface of the lower face of said chamber (8) is more hydrophilic than the surface of said at least one inlet (7), said hydrophilicity being measured by contact angle method.

2. Microfluidic device (4) according to claim 1, said microfluidic device (4) further containing at least:an outlet (5) located in said layers (1) and (2); anda well (6) located in said layers (1) and (2) which comprises said at least one inlet (7) and said chamber (8),said chamber (8) optionally comprising an injection port (13),said outlet (5) and said chamber (8) being in fluid communication through the side (8b) of the chamber (8).

3. Microfluidic device (4) according to claim 1 or 2, said microfluidic device (4) comprising:- at least a first hydrophilic spot (11) placed at the lower surface of said chamber (8), said at least first hydrophilic spot (11) extending contiguously towards the lower surface of said inlet (7) so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),said first hydrophilic spot (11) which form a 3D pattern wetting:■ being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2- hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or■ being etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8);and / or- a hydrophobic spot (18) placed at the lower surface of said inlet (7) so as to promote the confinement of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),said hydrophobic spot (18):■ being made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; or■ being a superhydrophobic surface, structured with micro / nano- texturization on the lower surface of said inlet (7).

4. Microfluidic device (4) according to any of claims 1 to 3, said microfluidic device (4) further comprising a second hydrophilic spot (IE) placed at the top surface of said chamber (8),said second hydrophilic spot (IE):- being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or- being etched surfaces structured with a laser or a plasma on the top surface of said chamber (8).

5. Microfluidic device (4) according to any of claims 1 to 4, wherein said first hydrophilic spot (11) being placed at the lower surface of said chamber (8) and / or said second hydrophilic spot (11 ’) placed at the top surface of said chamber (8) is(are) arranged in a 50-to-100-micron depth cavity(ies) engraved inside said lower surface of said chamber (8) and / or said top surface of said chamber (8).

6. Microfluidic device (4) according to any of claims 1 to 5, said microfluidic device (4) further comprising a third hydrophilic spot (101) placed at the surface of said side (8c) and a fourth hydrophilic spot (1001) placed at the surface of said side (8d),said third hydrophilic spot (101) and said fourth hydrophilic spot (1001):- being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or- being etched surfaces structured with a laser or a plasma on the surfaces of said sides (8c, 8d).

7. Microfluidic device (4) according to any of claims 1 to 6, wherein said outlet (5) and said chamber (8) are in fluid communication through the side (8b) of the chamber (8) by a channel (10) located in layer (2).

8. Microfluidic device (4) according to any of claims 1 to 7, wherein said outlet (5) comprises a cured porous polymer network layer (50),in particular said cured porous polymer network layer (50) being made of chitosan, polyethylene glycol diacrylate (PEGDA), polyethylene glycol monomethacrylate (PEGMA), porous polydimethylsiloxane (porous PDMS), Cellulose, polyvinylidene difluoride (PVDF), Polycarbonate (PC), Polysulfone (PS), Polyethersulfone (PES), Nylon, PTFE membranes or other air-permeable polymers with minimal shrinkage upon curing.

9. Microfluidic device (4) according to any of claims 1 to 8, wherein said microfluidic device (4) further comprises an inlet (9) located in layers (1) and (2),said outlet (5), said chamber (8) and said inlet (9) being in fluid communication through a channel (10) located in layer (2), said channel (10) being connected to said chamber (8) through the side (8b) of the chamber (8).

10. Microfluidic device (4) according to any of claims 1 to 9, said chamber (8) being divided into two adjacent compartments (14, 14’), aligned with the axis made from opposite sides (8a, 8b) and separated with a permeable engraved line (15), wherein each compartment (14, 14’) can trap the same or a different liquid hydrogel precursor solution (12, 12’) liable to be in situ polymerized and comprises:- said at least first hydrophilic spot (11) and said additional hydrophilic surface (Ila), and / or said hydrophobic spot (18); and- optionally said second hydrophilic spot (IE),and wherein each compartment (14’, 14’) optionally comprises an injection port (13, 13’).

11. Microfluidic device (4) according to any of claims 1 to 10, said microfluidic device (4) comprises at least two independent chambers (8, 80), said chambers (8, 80):- optionally comprising an injection port (13, 130);- being adjacent to its own inlet (7, 70);- being both in fluid communication with said outlet (5); and- comprising at least said first hydrophilic spot (11, 110) and said additional hydrophilic surface (Ila, 110a), and / or said hydrophobic spot (18, 180), and optionally said second hydrophilic spot (IF, 110’),wherein said chambers (8, 80) can trap the same or a different liquid hydrogel precursor solution (12, 120) liable to be in situ polymerized.

12. Microfluidic device (4) according to any of claims 1 to 11, wherein said microfluidic device (4) further comprises a baseless well-plate (16) stacked on the top layer (1), said baseless well-plate (16) comprising apertures (17) positioned above said inlet(s) (7, 9) and above said outlet (5) located in layers (1) and (2).

13. Microfluidic device (4) according to any of claims 1 to 12, wherein said microfluidic device (4) further comprises a liquid hydrogel precursor solution (12) liable to be in situ polymerized.

14. Microfluidic device (4) according to any of claims 1 to 13, wherein said microfluidic device (4) further comprises an in situ polymerized hydrogel (12a).

15. Microfluidic device (4) according to 13 or 14, wherein said liquid hydrogel precursor solution (12) liable to be in situ polymerized or said in situ polymerized hydrogel (12a) comprise: prokaryotic cells, eukaryotic cells or a mixture thereof.

16. Microfluidic device (4) according to claim 13 or 14, wherein:- said outlet (5) and said well (6) are filled with a medium, said medium diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8); or- said outlet (5), said well (6) and optionally said channel (10) are filled with a medium, said medium diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8); or- said well (6) is filled with a first medium, and said outlet (5), optionally said channel (10) and optionally said inlet (9) are filled with a second medium, said first and second media diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8),in particular said medium comprising a compound, said compound being preferably a drug,in particular said first medium or said second medium comprising a compound, said compound being preferably a drug.

17. Microfluidic device (4) according to any of claims 1 to 16, said microfluidic device (4) being multiplexed.

18. Process of manufacturing a microfluidic device (4) according to any of claims 1 to 17, said process comprising at least the following steps ofb. manufacturing of three layers using laser cutting to obtain: a top layer (1), a microfluidic middle layer (2) and a bottom layer (3);c. manufacturing a hydrophilic spot (11) to the top of said bottom layer (3) and optionally a hydrophilic spot (11’) to the bottom of said top layer (1) using:■ either a coating made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), polyethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof,■ or laser, or plasma, to obtained etched surfaces structured,to obtain a top layer (1) optionally comprising a hydrophilic spot (IE) and a bottom layer (3) comprising a hydrophilic spot (11),and / ormanufacturing a hydrophobic spot (18) to the top of said bottom layer (3) using:■ either a coating made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer,■ or a superhydrophobic surface, structured with micro / nano-texturization, to obtain a top layer (1) comprising a hydrophobic spot (18); andd. assembling and bounding said top layer (1) optionally comprising a hydrophilic spot (11’), said microfluidic middle layer (2) and said bottom layer (3) comprising a hydrophilic spot (11) and / or a hydrophobic spot (18) to obtain said microfluidic device (4) according to any of claims 4 to 17.

19. Process of manufacturing according to claim 18, said process further comprising a step of injection of a liquid hydrogel precursor solution (12) into a chamber (8) through an inlet (7), said liquid hydrogel precursor solution (12) polymerizing in situ, to obtain a microfluidic device (4) according to any of claims 8 to 10,in particular said liquid hydrogel precursor solution (12) comprising: prokaryotic cells, eukaryotic cells or a mixture thereof.

20. Use of a hydrophilicity difference to implement a 3D pattern wetting so as to trap a liquid hydrogel precursor solution (12) liable to be in situ polymerized within a microfluidic device (4) according to any of claims 1 to 17.

21. Use of a hydrophilicity difference according to claim 20, wherein:- at least a first hydrophilic spot (11) is placed at the lower surface of said chamber (8) and extends contiguously towards the lower surface of said inlet (7) so as to form an additional hydrophilic surface (Ila) to promote the suction of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),said first hydrophilic spot (11):■ being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or■ being etched surfaces structured with a laser or a plasma on the lower surface of said chamber (8);and / or- a hydrophobic spot (18) is placed at the lower surface of said inlet (7) so as to promote the confinement of said liquid hydrogel precursor solution (12) liable to be in situ polymerized from said inlet (7) to said chamber (8),said hydrophobic spot (18):■ being made of hydrophobic materials such as trichloro / perfluoroalkyl silane, fluoropolymer; or■ being a superhydrophobic surface, structured with micro / nano- texturization on the lower surface of said inlet (7).

22. Use of a hydrophilicity difference according to claim 20 or 21, wherein a second hydrophilic spot (IT) is placed at the top surface of said chamber (8),said second hydrophilic spot (IT):- being made of hydrophilic materials such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), Polydopamine (PDA), polyvinyl alcohol (PVA), poly(ethylene glycol) methacrylate (PEGMA), poly(2-hydroxyethyl methacrylate) (PHEMA), desiccated (dried) cell culture medium or a mixture thereof; or- being etched surfaces structured with a laser or a plasma on the top surface of said chamber (8).

23. Use of a microfluidic device according to any of claims 1 to 17 for in vitro cell culture and / or compound screening,in particular said cell being chosen among: prokaryotic cells, eukaryotic cells and a mixture thereof,in particular said compound being a drug.

24. Method for generating one diffusive compound gradient in a chamber (8) located in a microfluidic device according to any of claims 9 to 17, said method comprising at least the following steps of:a. filling a well (6) with a first medium comprising said compound, said first media comprising said compound diffusing in an in situ polymerized hydrogel (12a) located in said chamber (8) through a side (8a); andb. filling an inlet (9), which is in fluid connection with an outlet (5) and a channel (10), with a second medium lacking said compound, said second medium lacking said compound diffusing in said in situ polymerized hydrogel (12a) located in said chamber (8) through a side (8b),in particular said compound being a drug.

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