MICROFLUIDIC DEVICE FOR GENERATING COMBINATORIAL SPATIALLY CONTROLLABLE DIFFUSIVE GRADIENTS

FIELD OF THE INVENTION

The present invention relates to a microfluidic device for generating combinatorial spatially controllable diffusive gradients 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.

PRIOR ART

Evaluation of cell response to drugs is of great importance in precision medicine. In personalized cancer therapy, extensive screening of patient-derived primary tumor samples can lead to drug repurposing or discovery (Gorshkov K, et al. Advancing precision medicine with personalized drug screening. Vol. 24, Drug Discovery Today. Elsevier Ltd; 2019. p. 272- 8). In the case of infectious diseases, assessment of the antimicrobial susceptibility of bacterial isolates is necessary to detect resistance and/or to validate the selected empirical antibiotics (Jorgensen JH, Ferraro MJ. Vol. 49, Clinical Infectious Diseases. 2009. p. 1749-55). Yet three main shortcomings in current drug screening testing hamper the advancement of the field: long duration of the assays, low throughput, and lack of method to systematically test drug-drug interactions.

The time issue: The current standard antibiotic susceptibility tests (AST) are broth microdilution and agar disk diffusion. The duration of each assay is typically between 16-24 hours. However, considering the high number of cells required to conduct these tests, the total time for the overnight preculture, preparation, and susceptibility test is up to 72 hours. On the other hand, seeking early intervention, clinicians start an empirical therapy with the routine broad-spectrum antibiotics before receiving the test results (Shehab N, etal. 2013-2014. JAMA - Journal of the American Medical Association. 2016 Nov 22;316(20):2115-25). False speculation about the susceptibility of the infectious bacteria might result in prolonged hospitalization. Although recently some solutions reduced the considerable time needed to conduct the AST (Nguyen A v., et al. Analytical Chemistry. 2021 Apr 13;93(14):5789-96; Choi J, et al. Science Translational Medicine, 17 Dec 2014, Vol 6, Issue 267, p. 267ra174; Liu Z, et al. ChemPlusChem. 2017 May 1 ;82(5):792-801)), none provides a solution for both the throughput and drug-drug interactions problems.

The throughput issue: Routine antibiotic panels for susceptibility testing such as NMIC408 (product no. 448877, BD) and PMIC-90 (product no. 448439, BD) contain 20 to 30 antibiotics. Despite the advances in microfluidic technology and automation of liquid manipulations, most of the available technologies for drug screening rely on methods that are not suitable for high- throughput screening. For example, manual cell loading in each discrete chamber can severely reduce the multiplexing potential of a microfluidic device for rapid AST (Choi J, et al. Science Translational Medicine, 17 Dec 2014, Vol 6, Issue 267, p. 267ra174; Juskova P, et al. ACS Sensors. 2021 Jun 25;6(6):2202-10). Therefore, increasing throughput in such devices directly increases the labor. Moreover, large amounts of labor result in prolonged preparation time, which contradicts the rapidness claim of these methods.

The drug-drug interactions issue: understanding interactions of drug combinations can reveal important insight into the kinetics and other features of drug combinations (Richards R, et al. Nature Chemical Biology. 2020 Jul 1 ;16(7): 791-800). Regardless of the synergistic or antagonistic interaction of a drug combination, the outcome can contribute to designing novel therapies and controlling cellular states. The effectiveness of antibiotic combinations pushes the medical associations to recommend them even while they are still in the clinical development phase. For example, the guideline of Infectious Disease Society of America (IDSA) recommends aztreonam/avibactam for MBL-producing carbapenem-resistant enterobacterales due to the limited therapeutic options. The benefits of drug combinations are not limited to infectious disease or cancer. In 2016, Orkambi®, the combination of two FDA- approved drugs (lumacaftor and ivafactor), was approved as a treatment option for cystic fibrosis, the most frequent recessive genetic disorder in European populations (Gorshkov K, et al. Advancing precision medicine with personalized drug screening. Vol. 24, Drug Discovery Today. Elsevier Ltd; 2019. p. 272-8). The current method of performing drug combination studies is to manually prepare the combination at various ratios and dilutions in microtitration plates. Although robotic liquid manipulation considerably reduces the burden, further increase in throughput is essential. The number of test conditions for the pairwise combinatorial drug screening multiplies by the size of the drug library. For example, to test 100 drugs versus each other, 10,000 test conditions need to be tested. In addition, drug-drug interactions are concentration-dependent, meaning that a particular drug combination might show synergistic, antagonistic, or additive responses based on the concentration of each drug in the mixture. Thus, if it is considered ten concentrations for each drug in a combination, 1 ,000,000 test conditions need to be investigated. Even with the help of robots and multiplexing and miniaturization of microtitration plates, the cost of reagents, plastics, and infrastructure to handle the cultures is remarkably high (Agresti JJ, et al. Proceedings of the National Academy of Sciences of the United States of America. 2010 Mar 2;107(9):4004-9.). This example indicates a further need for multiplexing, miniaturization, and novelty to achieve satisfactory methods. Microfluidics can provide a tool to reach the required level of throughput. However, the existing microfluidics to study drug combinations are limited to either low-throughput assays (Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods. In: Clinical Microbiology Procedures Handbook. ASM Press; 2016. p. 5.16.1- 5.16.23.; Locascio L, Atencia-Fernandez FJ. Method and device for generating diffusive gradients in a microfluidic chamber. United States; US 8.216,526 B2, 2012) or the high throughput methods that involve complicated workflow and extensive analysis (Kulesa A, et al.. Proceedings of the National Academy of Sciences of the United States of America. 2018 Jun 26; 115(26)).

To address all these limitations, the inventors have developed the hereafter new microfluidic device.

BRIEF DESCRIPTION

The present invention relates to a multiplexed, miniaturized, and high-throughput geometry and device capable of generating simultaneous spatially crossing chemical gradients inside a porous medium such as a photopatterned hydrogel scaffold or a seedable porous scaffold, and their parallelization. The microfluidic device is composed of an array of multiple chambers, where crossing gradients are generated within each chamber, thereby allowing one to test the dose-response of pairwise combinations. In particular, within each chamber, the two gradients are generated thanks to 3 inlets: 1 for each of the 2 drugs and 1 for the medium. Gradients are independent of each other and can be maintained indefinitely constant. The mechanism of transport of molecules in the hydrogel is diffusion. Therefore, after a transient period for the formation of the gradient, which typically happens within minutes, the gradient is controlled by the geometry and is independent of the diffusive properties of the solutes, including their molecular weight. The microfluidic device can be multiplexed to create an array of compartmentalized photopatterned hydrogels, within which pairwise combinatorial chemical gradients of multiple test agents can be generated. At the level of the overall device, only 2N+1 inlets are required to test combinations in NxN chambers. The microfluidic device can be used to evaluate the susceptibility of bacteria (or other microorganisms or cells or any biological agent that can be embedded in the porous medium) to combinations of chemicals (e.g. drugs) at multiple doses. Moreover, the microfluidic device can be used to culture eukaryotic cells in a physiologically relevant 3D microenvironment, and to screen libraries of approved drugs or drug candidates for infectious disease, cancer, etc.

In view of the above, a first subject matter of the invention is therefore a microfluidic device for generating combinatorial spatially controllable diffusive gradients. A second subject matter of the invention concerns the processes of manufacturing the microfluidic device according to the invention. Further subject matters of the invention relate to uses and methods that can be implemented with the microfluidic device of the invention.

DETAILED DESCRIPTION

According to a first aspect, the subject matter of the invention concerns a microfluidic device for generating diffusive gradients comprising at least one assay chamber (20, 30; 200, 300), placed on a support (10), comprising: at least two zones (2, 3) substantially parallel to said support (10): a first zone (2) located below a second zone (3); one cell (21 ; 210) substantially parallel to said support (10), located in the first zone (2); and three independent channels (28, 31a, 41a; 250a, 260a, 301a) substantially parallel to said support (10): a first channel (28; 301a), a second channel (31a; 250a) and a third channel (41a; 260a), which are in fluid communication with the cell (21 ; 210) via three distinct contact point sets (x, y, z), each channel (28, 31a, 41a; 250a, 260a, 301a) being spread with only one of said zones (2, 3), and said zones (2, 3) comprising at least one channel, wherein the channel(s) located in the second zone (31a, 41a; 301a) are in fluid communication with said cell (21 ; 210) via through-hole(s) (23, 24; 230), said through-hole(s) (23, 24; 230) being located in the first zone (2) below said channel(s) (31a, 41a; 301a), adjacent to said cell (21 ; 210) and crossing substantially perpendicularly said first zone (2), said microfluidic device also comprising means for supplying said channels (28, 31a, 41a; 250a, 260a, 301a) with liquids (see Figures 1, 2).

Concerning the references mentioned between the brackets, it has to be understood that the is used to separate embodiments from each other, while is used to separate features within an embodiment. For instance, “three independent channels (28, 31a, 41a; 250a, 260a, 301a)” should be read as features 28, 31a and 41a belonging to the same embodiment, the one presented in Figure 1 , whereas features 250a, 260a and 301a belong to the same embodiment, the one presented in Figure 2.

By “diffusive gradient”, it means that the circulation of liquids, especially those containing and carrying test agents, in the microfluidic device of the invention generates a gradient of concentration of said test agents in the cell (21 ; 210). By “gradient of concentration”, it means the concentration of said test agents in the cell (21 ; 210) varies from one point to another of said cell (21 ; 210) (see Figure 15).

By “assay chamber”, it means the compartment resulting from the stacking of at least two zones (2, 3) on the support (10) and comprising all the components necessary (/.e. cell, channels and through-holes) for setting up at least one diffusive gradient in the cell (21 ; 210). By “at least one assay chamber”, it means the microfluidic device of the invention may comprise one assay chamber or several (see infra the ‘m x n’ embodiments).

By “zone” it means a part or a layer of the microfluidic device of the invention in which there is at least one component necessary (/.e. cell and/or channels and/or through-holes) for setting up at least one diffusive gradient such as a channel, a through-hole or the cell (21 ; 210). By “at least two zones”, it means the microfluidic device of the invention may comprise two zones or several such as 3 or 4 zones (see infra). In the context of the invention, said zones are manufactured:

■ either with materials that do not allow UV to pass (or any wavelength that activates the polymerization reaction of a photocurable hydrogel scaffold (22; 220) - see infra) including but not limited to: o glasses such as N-SF 11 ; o PMMA grades such as 0F302 (manufactured by Rohm); o Polycarbonate grades such as Panlite® L-1225Z; and/or o UV passing substrates coated with UV blockers such as lignin,

■ or with materials that let UV through (or any wavelength that activates the polymerization reaction of a photocurable hydrogel scaffold (22; 220) - see infra) including but not limited to:

- Glasses, such as soda lime and borofloat 33;

- Poly(methyl methacrylate) (PMMA) grades, such as 0F301 (manufactured by Rohm) and mcs-foil 148 (manufactured by Microfluidic ChipShop);

- Polydimethylsiloxane (PDMS);

- cyclic olefin copolymer (COC); and/or

- polystyrene (PS), knowing that said at least two zones (2, 3) can be manufactured with the same type of material or with different types of materials.

By “said at least two zones (2, 3) can be manufactured with the same type of materials”, it means that:

■ said at least two zones (2, 3) are all manufactured with materials belonging to the class of those that do not allow light {e.g. UV) to pass, said materials being liable to be different {e.g. zone (2) manufactured with N-SF11 and zone (3) manufactured with PMMA grades 0F302); or

■ said at least two zones (2, 3) are all manufactured with materials belonging to the class of those that allow light e.g. UV) to pass, said materials being liable to be different {e.g. zone (2) manufactured with PDMS and zone (3) manufactured with COC).

By “said at least two zones (2, 3) can be manufactured with different types of materials”, it means that at least one zone {e.g. 2) is manufactured with a material belonging to the class of those that do not allow light {e.g. UV) to pass, and at least one zone {e.g. 3) is manufactured with a material belonging to the class of those that allow light {e.g. UV) to pass. On this point and depending on the materials chosen, attention should be paid to the configuration of the microfluidic device of the invention, particularly the path of the channels {cf. infra), and vice versa.

By “path of the channels”, it means the path that a channel makes in a given zone, which could be for instance straight, L-shaped, U-shape, etc.

By “the stacking of at least two zones”, it means a first zone (2) is located below a second zone (3), said first zone (2) and second zone (3) being fixed together, i.e. covalently bounded to each other, in particular by plasma-activated bonding. Consequently, the first zone (2) corresponds to the lower zone and the second zone (3) corresponds to the upper zone.

By “support”, it means any material that supports an object placed on it. In the context of the invention, said support is manufactured with materials that let UV through (or any wavelength that activates the polymerization reaction of a photocurable hydrogel scaffold (22; 220) - see infra) including but not limited to: glass and transparent polymers such as cyclic olefin copolymer (COC).

By “assay chamber placed on a support”, it means the stack of at least two zones (2, 3), which makes it possible to define at least one assay chamber (20, 30; 200, 300), is laid on a support (10) and covalently bonded to said support (10) so that the surface of said support (10) in contact with said stack corresponds to the lower surface of the cell (21 ; 210) in which the gradient(s) are generated. In other words, it is the association of the support (10) with the stack that defines the hollow compartment corresponding to the cell (21 ; 210) of one assay chamber (20, 30; 200, 300).

By “cell”, it means the hollow compartment in which the gradient(s) are generated whose space is defined by the association of the support (10) with the stacking of at least two zones (2, 3).

By “channel” (or microchannel), it means a hollow micropipe with an inlet (also called fluidic input - see Figures) and an outlet (also called fluidic input - see Figures), or vice versa, through which a fluid can flow, the width:height ratio being comprised from 0.2 to 20, in particular from 1 to 10. By “independent channels”, it means the different channels or microchannels of the microfluidic device of the invention do not meet, do not intersect and have no overlap. By “three independent channels”, it means the microfluidic device of the invention comprises three different channels, each allowing said cell (21 ; 210) to be independently supplied with liquid, each channel being able to contain a same or different liquid comprising or not same or different test agents. In this way, it is possible to generate at least one, in particular at least two, diffusive gradients in the cell (21 ; 210).

By “fluid communication”, it means that a liquid can flow between at least two distinct components of the microfluidic device of the invention with the proviso that these two elements have necessary means for the circulation of said liquid. In other words, if:

- a channel (e.g. 28; 250a) is in fluid communication with the cell (21 ; 210), it means a liquid can flow between said channel and said cell (or a liquid can flow from said channel to said cell); - a through-hole (e.g. 23; 230) is in fluid communication with the cell (21 ; 210), it means a liquid can flow between said through-hole and said cell (or a liquid can flow from said through-hole to said cell); or

- a channel {e.g. 41a; 301a) is in fluid communication with a through-hole e.g. 23; 230) which is in fluid communication with the cell (21 ; 210), it means a liquid can flow from said channel to said cell via said through-hole.

In the invention, as the circulation of liquids has been designed to occur only in the components connected with I connected to the cell (21 ; 210) for generating diffusive gradient(s) in said cell (21 ; 210), it is understood that the stacking of at least two zones (2, 3) laid on the support (10) is made watertight/waterproof. This means that there is no liquid leakage at the junctions between the connected components. This also means that there is no liquid flowing out of the paths dedicated to the circulation of said liquids. By “paths dedicated to the circulation of said liquids”, it means the paths which go from an inlet of a channel to its outlet, which channel allows the liquid to reach the cell (21 ; 210) either directly or via a through-hole.

By “contact point set”, it means an opening, more or less extensive, located in the border of the cell (21 ; 210) which corresponds to the junction between said cell (21 ; 210) and a channel (e.g. 28; 250a) or between said cell (21 ; 210) and a through-hole (23; 24; 230), said junction allowing the passage of a liquid through it. By “distinct contact point sets”, it means that the contact point set ‘x’, the contact point set ‘y’ and the contact point set ‘z’ do not meet, do not intersect and have no overlap.

By “through-hole”, it means a hollow piece which connects and ensures fluid communication between two distinct components distributed in distinct zones, the width:height ratio being comprised from 0.1 to 10, in particular from 1 to 5. However, although the assembly of a channel with a through-hole, itself assembled with said cell allows the circulation of a fluid in these components, their assembly is not a channel according to the invention. It has also to be pointed out that functional difference between channels and through-holes is that microchannels carry fluid within a zone whereas through-holes carry fluid between two zones. By “through-hole [...] adjacent to said cell”, it means the through-hole is next to the said cell and these components are in fluid communication. By “through-hole [...] crossing substantially perpendicularly one zone”, it means the through-hole is located in the thickness (height) of the zone through which it passes.

By “microfluidic device also comprising means for supplying said channels (28, 31a, 41a; 250a, 260a, 301a) with liquids”, it means that despite the stacking of the zones (or layers) one on top of the other, with the upper zone overlapping the lower zone, the upper zone(s) moreover comprise(s) means {e.g. a cut-out) of access to the inlets and outlets (or vice versa) of the channel(s) located in the lower zone(s) so as to be able to load or unload therein the liquid(s) necessary for the implementation of the invention (see Figure 13). In other words, the subject matter of the invention concerns the microfluidic device as described above for generating diffusive gradients comprising at least one assay chamber (20, 30; 200, 300), placed on a support (10), comprising: at least two zones (2, 3) substantially parallel to said support (10): a first zone (2) located below a second zone (3); one cell (21 ; 210) substantially parallel to said support (10), located in the first zone (2); and three independent channels (28, 31a, 41a; 250a, 260a, 301a) substantially parallel to said support (10): o a first channel (28; 301a) comprising an inlet (25a; 301 b) and an outlet (25b; 301c), o a second channel (31a; 250a) comprising an inlet (31b; 250b) and an outlet (31c; 250c), and o a third channel (41a; 260a) comprising an inlet (41 b; 260b) and an outlet (41c; 260c), said three independent channels (28, 31a, 41a; 250a, 260a, 301a) being in fluid communication with the cell (21 ; 210) via three distinct contact point sets (x, y, z), each channel (28, 31a, 41a; 250a, 260a, 301a) being spread in only one of said zones (2, 3) and said zones (2, 3) comprising at least one channel, wherein the channel(s) located in the second zone (31a, 41a; 301a) are in fluid communication with said cell (21 ; 210) via through-hole(s) (23, 24; 230), said through-hole(s) (23, 24; 230) being located in the first zone (2) below said channel(s) (31a, 41a; 301a), adjacent to said cell (21 ; 210) and crossing substantially perpendicularly said first zone (2), said microfluidic device also comprising means for supplying said channels (28, 31a, 41a; 250a, 260a, 301a) with liquids, said means being cut-offs made in said zones (2, 3) located in line with the inlets (25a, 31 b, 41b; 301 b, 250b, 260b) and outlets (25b, 31c, 41c; 301c, 250c, 260c) of each of said channels (28, 31a, 41a; 250a, 260a, 301a).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein: the first zone (2) comprises one of said three independent channels (28) and the second zone (3) comprises two of said three independent channels (31a, 41a), the channels (31a) and (41a) being in fluid communication with said cell (21) via the through-holes (23) and (24) respectively (see Figure 1); or the first zone (2) comprises two of said three independent channels (250a, 260a) and the second zone (3) comprises one of said three independent channels (301a), the channel (301a) being in fluid communication with said cell (210) via the through-hole (230) (see Figure 2).

In another embodiment, the subject matter of the invention concerns a microfluidic device for generating diffusive gradients comprising at least one assay chamber (20, 30), placed on a support (10), said at least one assay chamber (20, 30) comprising: at least two zones (2, 3) substantially parallel to said support (10): a first zone (2) located below a second zone (3); and one cell (21) which is in fluid communication with: o a first channel (28) substantially parallel to said support (10) and located in said first zone (2), said first channel being a network of interconnected channels (26, 27, 28, 29); and o a second channel (31a) and a third channel (41a) substantially parallel to said support (10) and located in said second zone (3), wherein: the first zone (2) comprises: o said cell (21); o said network of interconnected channels (26, 27, 28, 29) which surrounds said cell (21), one of said interconnected channels (28) being adjacent and in fluid communication with a first contact point set (x) of said cell (21); o a first through-hole (24) adjacent and in fluid communication with a second contact point set (y) of said cell (21), and in fluid communication with said second channel (31a), said first through-hole (24) crossing substantially perpendicularly said first zone (2); and o a second through-hole (23) adjacent and in fluid communication with a third contact point set (z) of said cell (21), and in fluid communication with said third channel (41a), said second through-hole (23) crossing substantially perpendicularly said first zone (2), and the second zone (3) comprises: o said second channel (31a) which is in fluid communication with said cell (21) via said first through-hole (24); and o said third channel (41a) which is in fluid communication with said cell (21) via said second through-hole (23), said microfluidic device also comprising means for supplying said channels (26, 27, 28, 29, 31a, 41a) with liquids (see Figure 1).

In this set-up, it is understood that the implementation of the microfluidic device of the invention involves that: a first liquid is injected in the network of interconnected channels (26, 27, 28, 29) via the inlet (25a), said first liquid diffusing in the cell (21) via the contact point set (x); a second liquid is injected in the second channel (31a) via the inlet (31 b), said second liquid diffusing in the cell (21) via the contact point set (y) by using the through-hole (24) which makes the link between the cell (21) located in zone (2) and the channel (31a) located in zone (3); and a third liquid is injected in the third channel (41a) via the inlet (41b), said third liquid diffusing in the cell (21) via the contact point set (z) by using the through-hole (23) which makes the link between the cell (21) located in zone (2) and the channel (41a) located in zone (3), said first liquid can evacuate from the network of interconnected channels (26, 27, 28, 29) via the outlet (25b), said second liquid can evacuate from the second channel (31a) via the outlet (31c) and said third liquid can evacuate from the third channel (41a) via the outlet (41c). It should be noted, however, that inlets and outlets can be reversed. It should be also noted that injection of liquids into the microfluidic device of the invention can be made continuously (e.g. using microfluidic automation tools).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, said at least one assay chamber (20, 30, 40) further comprising a third zone (4) substantially parallel to said support (10) and located above the second zone (3), said third zone (4) comprising one of said three independent channels (41a), said channel (41a) being in fluid communication with said cell (21) via: a through-hole (32), said through-hole (32) being located in the second zone (3), crossing substantially perpendicularly said second zone (3) so as to be in fluid communication with said through-hole (23) and said channel (41a); and the through-hole (23) being located below the through-hole (32) (see Figures 3-6). In another embodiment, the subject matter of the invention concerns a microfluidic device for generating diffusive gradients comprising at least one assay chamber (20, 30, 40) placed on a support (10), said at least one assay chamber (20, 30, 40) comprising: at least three zones substantially parallel to said support (10): a lower zone (2), a middle zone (3) and an upper zone (4); and one cell (21) which is in fluid communication with: o a first channel (28) substantially parallel to said support (10) and located in said lower zone (2), said first channel being a network of interconnected channels (26, 27, 28, 29); o a second channel (31a) substantially parallel to said support (10) and located in said middle zone (3); and o a third channel (41a) substantially parallel to said support (10) and located in said upper zone (4), wherein: the lower zone (2) comprises: o said cell (21); o said network of interconnected channels (26, 27, 28, 29) which surrounds said cell (21), one of said interconnected channels (28) being adjacent and in fluid communication with a first contact point set (x) of said cell (21); o a first through-hole (24) adjacent and in fluid communication with a second contact point set (y) of said cell (21), and in fluid communication with said second channel (31a), said first through-hole (24) crossing substantially perpendicularly said lower zone (2); and o a second through-hole (23) adjacent and in fluid communication with a third contact point set (z) of said cell (21), and in fluid communication with a third through-hole (32) located in the middle zone (3), said second through-hole (23) crossing substantially perpendicularly said lower zone (2), the middle zone (3) comprises: o said second channel (31a) which is in fluid communication with said cell (21) via said first through-hole (24); and o said third through-hole (32), which is in fluid communication with said cell (21) via said second through-hole (23) and in fluid communication with said third channel (41a), said third through-hole (32) crossing substantially perpendicularly said middle zone (3), and the upper zone (4) comprises: o said third channel (41a) which is in fluid communication with said cell (21) via both said third through-hole (32) and second through-hole (23), said microfluidic device also comprising means for supplying said channels (26, 27, 28, 29, 31a, 41a) with liquids (see Figures 3-6).

In this set-up, it is understood that the implementation of the microfluidic device of the invention involves that: a first liquid is injected in the network of interconnected channels (26, 27, 28, 29) via the inlet (25a), said first liquid diffusing in the cell (21) via the contact point set (x); a second liquid is injected in the second channel (31a) via the inlet (31 b), said second liquid diffusing in the cell (21) via the contact point set (y) by using the through-hole (24) which makes the link between the cell (21) located in zone (2) and the channel (31a) located in zone (3); and a third liquid is injected in the third channel (41a) via the inlet (41b), said third liquid diffusing in the cell (21) via the contact point set (z) by using the through-hole (32) which makes the link between the through-hole (23) located in zone (2) and the channel (41a) located in zone (4) and the through-hole (23) which makes the link between the cell (21) located in zone (2) and the through-hole (32) located in zone (3), said first liquid can evacuate from the network of interconnected channels (26, 27, 28, 29) via the outlet (25b), said second liquid can evacuate from the second channel (31a) via the outlet (31c) and said third liquid can evacuate from the third channel (41a) via the outlet (41c). It should be also noted that injection of liquids into the microfluidic device of the invention can be made continuously (e.g. using microfluidic automation tools).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said zones correspond to: separate layers of the at least one assay chamber, the stacking of which constitute said at least one assay chamber (20, 30; 200, 300; 20, 30, 40); or different parts of the at least one assay chamber which taken as a whole constitute said at least one assay chamber (20, 30; 200, 300; 20, 30, 40).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said support (10) is a transparent support. By “transparent support”, it means a support which allows light (in particular UV or any wavelength that activates the polymerization reaction of a photocurable hydrogel scaffold (22; 220) - see infra) to pass through and objects behind it to appear clearly.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said transparent support (10) comprises a removable photomask or a directly printed photomask into the support (10), said photomask comprising an optical window (11) allowing light (in particular UV or any wavelength that activates the polymerization reaction of a photocurable hydrogel scaffold (22; 220) - see infra) to pass through only at the cell level. By “photomask”, it means a mask which does not allow light to pass through.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said cell (21 ; 210) has a symmetric axis a-a’: on the left of which said through-hole (24) is located and on the right of which said through-hole (23) is located (see Figures 1, 3, 5, 7, 9 and 11); or on the right of which said through-hole (24) is located and on the left of which said through-hole (23) is located (see Figures 4, 6, 8, 10 and 12).

By “said cell (21 ; 210) has a symmetric axis a-a’”, it means the cell (21 ;210) located in the lower zone (2) is traversed in its plane by an axis passing through the contact point set (x), which axis is perpendicular to the line passing through the contact point sets (y) and (z).

In a particular embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said cell (21 ; 210) has a symmetric axis a-a’ on the left of which said through-hole (24) is located and on the right of which said through-hole (23) is located (see Figures 1, 3, 5, 7, 9 and 11). In this set-up, it is understood that due to the superposition of the zones and the connections between the zones, the elements connected to the through- hole (23), in particular the through-hole (32), are located on the right of the axis a-a’ and therefore on the right side of the cell (21 ; 210) and those connected to the through-hole (24) are located on the left of the axis a-a’ and therefore on the left side of the cell (21 ; 210).

In a particular embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said cell (21 ; 210) has a symmetric axis a-a’ on the right of which said through-hole (24) is located and on the left of which said through-hole (23) is located (see Figures 2, 4, 6, 8, 10 and 12). In this set-up, it is understood that due to the superposition of the zones and the connections between the zones, the elements connected to the through- hole (23), in particular the through-hole (32), are located on the left of the axis a-a’ and therefore on the left side of the cell (21 ; 210) and those connected to the through-hole (24) are located on the right of the axis a-a’ and therefore on the right side of the cell (21 ; 210).

As previously mentioned, said at least two zones (2, 3) can be light (e.g. UV) permissive or light {e.g. UV) blocking, and can be manufactured with:

■ the same type of material among those that do not allow light {e.g. UV) to pass;

■ the same type of material among those that allow light e.g. UV) to pass; or

■ with different types of materials, i.e. at least one zone {e.g. 2) made with a material selected from those that do not allow light {e.g. UV) to pass, and at least one zone {e.g. 3) made with a material selected from those that allow light {e.g. UV) to pass.

The microfluidic device of the invention can therefore be made in different ways depending on the material or materials chosen. Nevertheless, depending on the material(s) chosen, care must be taken with regard to the path of the channels in zone(s) above the zone (2) which comprises the cell (21 ; 210) so as not to compromise the implementation of the microfluidic device of the invention, in particular when it involves the use of a photocurable hydrogel scaffold (22; 220). Indeed, as the polymerisation of the photocurable hydrogel scaffold (22; 220) is only desired at the level of the cell (21 ; 210), it is necessary to avoid that it also polymerises in the channels, hence the use of a photomask, and of light-blocking materials and/or of a particular path of the channels so that they bypass the orthogonal projection of the surface of said cell (21 ; 210). As light {e.g. UV) comes from the bottom of the microfluidic device of the invention, the following should therefore be considered:

[1] the case where said at least two zones (2, 3) are all manufactured with light {e.g. UV) blocking material;

[2] the case where among said at least two zones (2, 3), only one zone is manufactured with light {e.g. UV) blocking material whereas the other(s) not; and

[3] the case where said at least two zones (2, 3) are all manufactured with light {e.g. UV) permissive material.

[1] In the case of a microfluidic device wherein said at least two zones (2, 3) are all manufactured with light (e.g. UV) blocking material

Here, as zone (2) blocks light so as to prevent it from reaching zone (3), the polymerisation of the photocurable hydrogel scaffold (22; 220) (with the help of the photomask) may only occur at the level of the cell (21 ; 210) located in zone (2). The path of the channels in zone (3) has thus no specific constraint and the channels may bypass (or not) the orthogonal projection of the surface of said cell (21 ; 210). This applies when the microfluidic device of the invention comprises at least two zones (2, 3) (see Figure 22) or at least three zones (2, 3, 4) (see Figure 23).

[2] In the case of a microfluidic device wherein among at least two zones (2, 3) only one zone is manufactured with light (e.g. UV) blocking material whereas the other(s) is (are) not Considering the microfluidic device of the invention implemented with at least two zones (2, 3): i. If it is the zone (2) which blocks light so as to prevent it from reaching zone (3), the polymerisation of the photocurable hydrogel scaffold (22; 220) (with the help of the photomask) may only occur at the level of the cell (21 ; 210) located in zone (2). The path of the channels in zone (3) has thus no specific constraint and the channels may bypass (or not) the orthogonal projection of the surface of said cell (21 ; 210) (see Figure 22). ii. If it is the zone (3) which blocks light, the polymerisation of the photocurable hydrogel scaffold (22; 220) (with the help of the photomask) may occur at the level of the cell (21 ; 210) located in zone (2) and at the level of the orthogonal projection of the surface of said cell (21 ; 210) located in the above zone (3). The path of the channels in zone (3) is thus constrained and the channels have to bypass the orthogonal projection of the surface of said cell (21 ; 210) (see Figures 1-2).

Considering the microfluidic device of the invention implemented with at least three zones (2, 3, 4): i. If it is the zone (2) which blocks light so as to prevent it from reaching zones (3, 4), the polymerisation of the photocurable hydrogel scaffold (22; 220) (with the help of the photomask) may only occur at the level of the cell (21 ; 210) located in zone (2). The path of the channels in zones (3, 4) has thus no specific constraint and the channels may bypass (or not) the orthogonal projection of the surface of said cell (21 ; 210) (see Figure 23). ii. If it is the zone (3) which blocks light so as to prevent it from reaching zone (4), the polymerisation of the photocurable hydrogel scaffold (22; 220) (with the help of the photomask) may occur at the level of the cell (21 ; 210) located in zone (2) and at the level of the orthogonal projection of the surface of said cell (21 ; 210) located in the above zone (3). The path of the channels:

- in zone (3) is thus constrained and the channels have to bypass the orthogonal projection of the surface of said cell (21 ; 210); and - in zone (4) has thus no specific constraint and the channels may bypass (or not) the orthogonal projection of the surface of said cell (21 ; 210) (see Figure 24). iii. If it is the zone (4) which blocks light, the polymerisation of the photocurable hydrogel scaffold (22; 220) (with the help of the photomask) may occur at the level of the cell (21 ; 210) located in zone (2) and at the level of the orthogonal projection of the surface of said cell (21 ; 210) located in both the above zone (3) and zone (4). The path of the channels in zones (3, 4) is thus constrained and the channels have to bypass the orthogonal projection of the surface of said cell (21 ; 210) (see Figures 3-12).

In view of the above, the following rule can be established: “Above the light (e.g. UV) blocking zone [hereafter LBZ], the path of the channel(s) is free of constraints, whereas the path of the channels located in the LBZ depends on the zone on which said LBZ rests. If said LBZ rests on another light (e.g. UV) blocking zone, the path of the channel(s) located in the LBZ is free of constraints; if said LBZ rests on a light (e.g. UV) permissive zone the path of the channel(s) located in the LBZ has to bypass the cell and its orthogonal projection.”

It has to be pointed out that with the implementation of the microfluidic device of the invention with at least three zones (2, 3, 4), it is also possible to have two zones manufactured with light (e.g. UV) blocking material. In case the zones (2) and (3) or the zones (2) and (4) are manufactured with light (e.g. UV) blocking material, this is equivalent to the situation described in i. In case the zones (3) and (4) which are manufactured with light (e.g. UV) blocking material, this is equivalent to the situation described in ii.

The above rule must be then qualified in cases where zones (2) and (4) are light blocking and zone (3) is light permissive, because this rule is then not true for zone (4). Indeed, in this case, zone (4) rests on a light-permissive zone and thus, the channels should bypass the orthogonal projection of the cell. However, as zone (2) is light blocking, there will not be any geometrical constraints for channels in zone (4). Consequently, if said LBZ rests on a light permissive zone but if anywhere under the light permissive zone, there is a light blocking zone, then the geometry of the channels in LBZ will not have any constraints.

It is thus understood that in another embodiment, the subject matter of the invention concerns the microfluidic device as described above for generating diffusive gradients comprising at least one assay chamber (20, 30; 200, 300), placed on a support (10), comprising: at least two zones (2, 3) substantially parallel to said support (10): a first zone (2) located below a second zone (3); one cell (21 ; 210) substantially parallel to said support (10), located in the first zone (2); and three independent channels (28, 31a, 41a; 250a, 260a, 301a) substantially parallel to said support (10): a first channel (28; 301a), a second channel (31a; 250a) and a third channel (41a; 260a), which are in fluid communication with the cell (21 ; 210) via three distinct contact point sets (x, y, z), each channel (28, 31a, 41a; 250a, 260a, 301a) being spread with only one of said zones (2, 3) and said zones (2, 3) comprising at least one channel, wherein the channel(s) located in the second zone (31a, 41a; 301a) are in fluid communication with said cell (21 ; 210) via through-hole(s) (23, 24; 230), said through-hole(s) (23, 24; 230) being located in the first zone (2) below said channel(s) (31a, 41a; 301a), adjacent to said cell (21 ; 210) and crossing substantially perpendicularly said first zone (2), wherein at least said first zone (2) is manufactured with a light (e.g. UV) blocking material, whereby path of the channels (31a, 41a; 301a) located in zone (3) which is located above said first zone (2) having no constraints, said microfluidic device also comprising means for supplying said channels (28, 31a, 41a; 250a, 260a, 301a) with liquids.

[31 In the case of a microfluidic device wherein said at least two zones (2, 3) are all manufactured with light (e.q. UV) permissive material

Here, in any case, the polymerisation of the photocurable hydrogel scaffold (22; 220) (with the help of the photomask) may occur at the level of the cell (21 ; 210) located in zone (2) and at the level of the orthogonal projection of the surface of said cell (21 ; 210) located in zone (3). The path of the channels in zone (3) is thus constrained and the channels have to bypass the orthogonal projection of the surface of said cell (21 ; 210). This applies when the microfluidic device of the invention comprises at least two zones (2, 3) (see Figures 1-2) or at least three zones (2, 3, 4) (see Figure 3-12).

In other word, the subject matter of the invention concerns the microfluidic device as described above, wherein all said three independent channels (28, 31a, 41a; 250a, 260a, 301a) are outside the orthogonal projection of the surface of said cell (21 ; 210) (see Figures 1-12). By “all said three independent channels are outside the orthogonal projection of the surface of said cell”, it means that despite the overlapping of the zones, none of the components in the upper zones (3 and/or 4) passes over the cell (21 ; 210) in the lowest zone (2). In this way, microscopic observation of the assay chamber (20, 30, 40) is not obstructed by any of the fluidic components of the microfluidic device of the invention.

Beyond these considerations, the following embodiments are independent of the nature of the material used:

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein: said first channel (31a) is substantially straight and said second channel (41a) is L- shaped or U-shaped so as to be outside the orthogonal projection of the surface of said cell (21) (see Figures 3, 4); or said first channel (31a) is L-shaped or U-shaped so as to be outside the orthogonal projection of the surface of said cell (21) and said second channel (41a) is substantially straight (see Figures 5, 6).

By “substantially straight”, it means that the channels which are qualified as being substantially straight have a substantially rectilinear form. By “L-shaped or U-shaped”, it means the channels which are qualified as being L-shaped or U-shaped have a substantially rectilinear form wherein there is at least one angle (e.g. at least one 90° angle) so that the channels have a L or U shape. It has to be pointed out using these particular geometric forms of the channels several assay chamber can be cleverly and efficiently multiplexed.

In a particular embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said first channel (31a) is substantially straight and said second channel (41a) is L-shaped or U-shaped so as to be outside the orthogonal projection of the surface of said cell (21) (see Figures 3, 4).

In a particular embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said first channel (31a) is L-shaped or U-shaped so as to be outside the orthogonal projection of the surface of said cell (21) and said second channel (41a) is substantially straight (see Figures 5, 6).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein both said first channel (31a) and second channel (41a) are substantially perpendicular to each other and substantially straight. In another embodiment, the subject matter of the invention concerns a microfluidic device for generating diffusive gradients comprising at least one assay chamber (20, 30, 40) placed on a support (10), said at least one assay chamber (20, 30, 40) comprising: at least three zones substantially parallel to said support (10): a lower zone (2), a middle zone (3) and an upper zone (4); and one cell (21) which is in fluid communication with: o a first channel (28) substantially parallel to said support (10) and located in said lower zone (2), said first channel being a network of interconnected channels (26, 27, 28, 29); o a second channel (31a) substantially parallel to said support (10) and located in said middle zone (3); and o a third channel (41a) substantially parallel to said support (10) and located in said upper zone (4), wherein: the lower zone (2) comprises: o said cell (21); o said network of interconnected channels (26, 27, 28, 29) which surrounds said cell (21), one of said interconnected channels (28) being adjacent and in fluid communication with a first contact point set (x) of said cell (21); o a first through-hole (24) adjacent and in fluid communication with a second contact point set (y) of said cell (21), and in fluid communication with said second channel (31a), said first through-hole (24) crossing substantially perpendicularly said lower zone (2); and o a second through-hole (23) adjacent and in fluid communication with a third contact point set (z) of said cell (21), and in fluid communication with a third through-hole (32) located in the middle zone (3), said second through-hole (23) crossing substantially perpendicularly said lower zone (2), the middle zone (3) comprises: o said second channel (31a) which is in fluid communication with said cell (21) via said first through-hole (24); and o said third through-hole (32), which is in fluid communication with said cell (21) via said second through-hole (23) and in fluid communication with said third channel (41a), said third through-hole (32) crossing substantially perpendicularly said middle zone (3), and the upper zone (4) comprises: o said third channel (41a) which is in fluid communication with said cell (21) via both said third through-hole (32) and second through-hole (23), wherein: both said first channel (31a) and second channel (41a) are outside the orthogonal projection of the surface of said cell (21); said cell (21; 210) has a symmetric axis a-a’ on the left of which said through-hole (24) is located and on the right of which said through-hole (23) is located; and said first channel (31a) is substantially straight and said second channel (41a) is L- shaped or U-shaped so as to be outside the orthogonal projection of the surface of said cell (21), said microfluidic device also comprising means for supplying said channels (26, 27, 28, 29, 31a, 41a) with liquids (see Figure 3).

In another embodiment, the subject matter of the invention concerns a microfluidic device for generating diffusive gradients comprising at least one assay chamber (20, 30, 40) placed on a support (10), said at least one assay chamber (20, 30, 40) comprising: at least three zones substantially parallel to said support (10): a lower zone (2), a middle zone (3) and an upper zone (4); and one cell (21) which is in fluid communication with: o a first channel (28) substantially parallel to said support (10) and located in said lower zone (2), said first channel being a network of interconnected channels (26, 27, 28, 29); o a second channel (31a) substantially parallel to said support (10) and located in said middle zone (3); and o a third channel (41a) substantially parallel to said support (10) and located in said upper zone (4), wherein: the lower zone (2) comprises: o said cell (21); o said network of interconnected channels (26, 27, 28, 29) which surrounds said cell (21), one of said interconnected channels (28) being adjacent and in fluid communication with a first contact point set (x) of said cell (21); o a first through-hole (24) adjacent and in fluid communication with a second contact point set (y) of said cell (21), and in fluid communication with said second channel (31a), said first through-hole (24) crossing substantially perpendicularly said lower zone (2); and o a second through-hole (23) adjacent and in fluid communication with a third contact point set (z) of said cell (21), and in fluid communication with a third through-hole (32) located in the middle zone (3), said second through-hole (23) crossing substantially perpendicularly said lower zone (2), the middle zone (3) comprises: o said second channel (31a) which is in fluid communication with said cell (21) via said first through-hole (24); and o said third through-hole (32), which is in fluid communication with said cell (21) via said second through-hole (23) and in fluid communication with said third channel (41a), said third through-hole (32) crossing substantially perpendicularly said middle zone (3), and the upper zone (4) comprises: o said third channel (41a) which is in fluid communication with said cell (21) via both said third through-hole (32) and second through-hole (23), wherein: both said first channel (31a) and second channel (41a) are outside the orthogonal projection of the surface of said cell (21); said cell (21 ; 210) has a symmetric axis a-a’ on the right of which said through-hole (24) is located and on the left of which said through-hole (23) is located; and said first channel (31a) is substantially straight and said second channel (41a) is L- shaped or U-shaped so as to be outside the orthogonal projection of the surface of said cell (21), said microfluidic device also comprising means for supplying said channels (26, 27, 28, 29, 31a, 41a) with liquids (see Figure 4).

In another embodiment, the subject matter of the invention concerns a microfluidic device for generating diffusive gradients comprising at least one assay chamber (20, 30, 40) placed on a support (10), said at least one assay chamber (20, 30, 40) comprising: at least three zones substantially parallel to said support (10): a lower zone (2), a middle zone (3) and an upper zone (4); and one cell (21) which is in fluid communication with: o a first channel (28) substantially parallel to said support (10) and located in said lower zone (2), said first channel being a network of interconnected channels (26, 27, 28, 29); o a second channel (31a) substantially parallel to said support (10) and located in said middle zone (3); and o a third channel (41a) substantially parallel to said support (10) and located in said upper zone (4), wherein: the lower zone (2) comprises: o said cell (21); o said network of interconnected channels (26, 27, 28, 29) which surrounds said cell (21), one of said interconnected channels (28) being adjacent and in fluid communication with a first contact point set (x) of said cell (21); o a first through-hole (24) adjacent and in fluid communication with a second contact point set (y) of said cell (21), and in fluid communication with said second channel (31a), said first through-hole (24) crossing substantially perpendicularly said lower zone (2); and o a second through-hole (23) adjacent and in fluid communication with a third contact point set (z) of said cell (21), and in fluid communication with a third through-hole (32) located in the middle zone (3), said second through-hole (23) crossing substantially perpendicularly said lower zone (2), the middle zone (3) comprises: o said second channel (31a) which is in fluid communication with said cell (21) via said first through-hole (24); and o said third through-hole (32), which is in fluid communication with said cell (21) via said second through-hole (23) and in fluid communication with said third channel (41a), said third through-hole (32) crossing substantially perpendicularly said middle zone (3), and the upper zone (4) comprises: o said third channel (41a) which is in fluid communication with said cell (21) via both said third through-hole (32) and second through-hole (23), wherein: both said first channel (31a) and second channel (41a) are outside the orthogonal projection of the surface of said cell (21); said cell (21 ; 210) has a symmetric axis a-a’ on the left of which said through-hole (24) is located and on the right of which said through-hole (23) is located; and said first channel (31a) is L-shaped or U-shaped so as to be outside the orthogonal projection of the surface of said cell (21) and said second channel (41a) is substantially straight, said microfluidic device also comprising means for supplying said channels (26, 27, 28, 29, 31a, 41a) with liquids (see Figure 5).

In another embodiment, the subject matter of the invention concerns a microfluidic device for generating diffusive gradients comprising at least one assay chamber (20, 30, 40) placed on a support (10), said at least one assay chamber (20, 30, 40) comprising: at least three zones substantially parallel to said support (10): a lower zone (2), a middle zone (3) and an upper zone (4); and one cell (21) which is in fluid communication with: o a first channel (28) substantially parallel to said support (10) and located in said lower zone (2), said first channel being a network of interconnected channels (26, 27, 28, 29); o a second channel (31a) substantially parallel to said support (10) and located in said middle zone (3); and o a third channel (41a) substantially parallel to said support (10) and located in said upper zone (4), wherein: the lower zone (2) comprises: o said cell (21); o said network of interconnected channels (26, 27, 28, 29) which surrounds said cell (21), one of said interconnected channels (28) being adjacent and in fluid communication with a first contact point set (x) of said cell (21); o a first through-hole (24) adjacent and in fluid communication with a second contact point set (y) of said cell (21), and in fluid communication with said second channel (31a), said first through-hole (24) crossing substantially perpendicularly said lower zone (2); and o a second through-hole (23) adjacent and in fluid communication with a third contact point set (z) of said cell (21), and in fluid communication with a third through-hole (32) located in the middle zone (3), said second through-hole (23) crossing substantially perpendicularly said lower zone (2), the middle zone (3) comprises: o said second channel (31a) which is in fluid communication with said cell (21) via said first through-hole (24); and o said third through-hole (32), which is in fluid communication with said cell (21) via said second through-hole (23) and in fluid communication with said third channel (41a), said third through-hole (32) crossing substantially perpendicularly said middle zone (3), and the upper zone (4) comprises: o said third channel (41a) which is in fluid communication with said cell (21) via both said third through-hole (32) and second through-hole (23), wherein: both said first channel (31a) and second channel (41a) are outside the orthogonal projection of the surface of said cell (21); said cell (21 ; 210) has a symmetric axis a-a’ on the right of which said through-hole (24) is located and on the left of which said through-hole (23) is located; and said first channel (31a) is L-shaped or U-shaped so as to be outside the orthogonal projection of the surface of said cell (21) and said second channel (41a) is substantially straight, said microfluidic device also comprising means for supplying said channels (26, 27, 28, 29, 31a, 41a) with liquids (see Figure 6).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, said at least one assay chamber (20, 30, 40) further comprising a fourth zone (5) substantially parallel to said support (10) and located above the third zone (4), said fourth zone (5) comprising an independent channel “j”, said channel “j” being in fluid communication with said cell (21) at a contact point set “k” (distinct from the contact point sets (x, y, z)) via: a through-hole “i", said through-hole “i" being located in the third zone (4), crossing substantially perpendicularly said third zone (4) so as to be in fluid communication with a through-hole “ii” and said channel “j”; the through-hole “ii”, said through-hole “ii” being located in the second zone (3), crossing substantially perpendicularly said second zone (3) so as to be in fluid communication with a through-hole “Hi” and said through-hole “i”; and the through-hole “Hi”, said through-hole “Hi” being located in the first zone (2), below the through-hole “ii”, adjacent to said cell (21 ; 210) and crossing substantially perpendicularly said first zone (2), said microfluidic device also comprising the means for supplying said channel “j” with liquid.

It has to be pointed out that this particular embodiment allows to perform a method for generating at least three diffusive gradients in the cell (21). In this set-up, it is also understood that the implementation of the microfluidic device of the invention involves that: a first liquid is injected in the network of interconnected channels (26, 27, 28, 29) via the inlet (25a), said first liquid diffusing in the cell (21) via the contact point set (x); a second liquid is injected in the second channel (31a) via the inlet (31 b), said second liquid diffusing in the cell (21) via the contact point set (y) by using the through-hole (24) which makes the link between the cell (21) located in zone (2) and the channel (31a) located in zone (3); a third liquid is injected in the third channel (41a) via the inlet (41b), said third liquid diffusing in the cell (21) via the contact point set (z) by using the through-hole (32) which makes the link between the through-hole (23) located in zone (2) and the channel (41a) located in zone (4) and the through-hole (23) which makes the link between the cell (21) located in zone (2) and the through-hole (32) located in zone (3); and a fourth liquid is injected in the fourth channel “j” via the inlet “j _a ”, said fourth liquid diffusing in the cell (21) via the contact point set “k” by using the through-hole “i” which makes the link between the through-hole “ii” located in zone (3) and the channel “j” located in zone (5), the through-hole “ii” which makes the link between the through-hole “iii” located in zone (2) and the through-hole “i” located in zone (4) and the through-hole “iii” which makes the link between the cell (21) located in zone (2) and the through-hole “ii” located in zone (3), said first liquid can evacuate from the network of interconnected channels (26, 27, 28, 29) via the outlet (25b), said second liquid can evacuate from the second channel (31a) via the outlet (31c), said third liquid can evacuate from the third channel (41a) via the outlet (41c) and said fourth liquid can evacuate from the fourth channel “j” via the outlet “j ”. It should be also noted that injection of liquids into the microfluidic device of the invention can be made continuously (e.g. using an microfluidic automation tools).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said cell (21 ; 210) is a 4-sidded cell, in particular a square cell. In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein both said through hole (24) and said through-hole (23) are located on adjacent sides of said cell (21).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein both said through hole (24) and said through-hole (23) are located on opposite sides of said cell (21).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said cell (21 ; 210) contains with a photocurable hydrogel scaffold (22; 220) or a seedable porous scaffold (22; 220).

By “hydrogel scaffold”, it means a three-dimensional network composed of hydrophilic polymers crosslinked either through covalent bonds or held together via physical intramolecular and intermolecular attractions (El-Sherbiny IM, et al. Glob Cardiol Sci Pract. 2013;2013(3):316-342. Published 2013 Nov 1.). By “photocurable hydrogel scaffold”, it means a hydrogel which obtains its structure/scaffold only after exposure to light.

By “seedable porous scaffold”, it means a three-dimensional network composed of inorganic material.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said photocurable hydrogel scaffold (22; 220) or said seedable porous scaffold (22; 220) is adapted for the growth of microorganisms in a 3D environment, said photocurable hydrogel scaffold (22; 220) or said seedable porous scaffold (22; 220) comprising at least one microorganism chosen among eukaryotic cell and procaryotic cell.

By “adapted for the growth of microorganisms in a 3D environment”, it means the photocurable hydrogel scaffold (22; 220) or the seedable porous scaffold (22; 220) used in the microfluidic device allows the in vitro culture of microorganisms. In other words, seeding cells in these materials does not affect the viability of microorganisms.

By “microorganism chosen among eukaryotic cell”, it means a cell of plant or animal origin, preferably a cell of animal origin such as human cells.

By “microorganism chosen among procaryotic cell”, it means a Bacteria cell or an Archaea cell. In a particular embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said microorganism is an eucaryotic cell. In a particular embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said microorganism is a procaryotic cell.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein: a first channel (e.g. 28; 301a) of said three independent channels allows a first liquid flows from an inlet (e.g. 25a; 301b) towards an outlet (e.g. 25b; 301c) and allows said first liquid to diffuse in said cell (21 ; 210); a second channel (e.g. 31a; 250a) of said three independent channels allows a second liquid comprising a test agent ‘A’ flows from an inlet (e.g. 31b; 250b) towards an outlet (e.g. 31c; 250c) and allows said second liquid comprising a test agent ‘A’ to diffuse in said cell (21 ; 210); and a third channel (e.g. 41a, 260a) of said three independent channels allows a third liquid comprising a test agent ‘B’ flows from an inlet (e.g. 41b; 260b) towards an outlet (e.g. 41c; 260c) and allows said third liquid comprising a test agent ‘B’ to diffuse in said cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein a fourth channel “j” allows a fourth liquid comprising a test agent ‘C’ flows from an inlet “j _a ” towards an outlet “j ” and allows said fourth liquid comprising a test agent ‘C’ to diffuse in said cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein: a first channel (e.g. 28; 301a) of said three independent channels is filled with a first liquid, said first liquid diffusing in said cell (21 ; 210); a second channel (e.g. 31a; 250a) of said three independent channels is filled with a second liquid comprising a test agent ‘A’, said second liquid comprising a test agent ‘A’ diffusing in said cell (21 ; 210); and a third channel (e.g. 41a, 260a) of said three independent channels is filled with a third liquid comprising a test agent ‘B’, said third liquid comprising a test agent ‘B’ diffusing in said cell (21 ; 210). In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein a fourth channel “j” is filled with a fourth liquid comprising a test agent ‘C’, said fourth liquid comprising a test agent ‘C’ diffusing in said cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said first liquid, second liquid and third liquid are culture media, said culture media being chosen among: Luria broth, Lennox broth, Mueller Hinton broth, M9, RPMI-1640 Medium, F12 Medium, Dulbecco's Modified Eagle Medium, etc. with or without additional components such as fetal bovine serum.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said first liquid, second liquid, third liquid and fourth liquid are culture media, said culture media being chosen among: Luria broth, Lennox broth, Mueller Hinton broth, M9, RPMI-1640 Medium, F12 Medium, Dulbecco's Modified Eagle Medium, etc. with or without additional components such as fetal bovine serum.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘A’ is chosen among: potentially therapeutic molecules such as small molecules (e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.y peptides such as cytokines (e.g. TNF-a, I L-1 b, IL-6, etc.

- proteins such as antibodies (e.g. Rituxan™, Herceptin™, Humira™, Benlysta™, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.y etc.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘B’ is chosen among: potentially therapeutic molecules such as small molecules (e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.y peptides such as cytokines (e.g. TNF-a, I L-1 b, IL-6, etc.y

- proteins such as antibodies (e.g. Rituxan™, Herceptin™, Humira™, Benlysta™, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.y etc.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘C’ is chosen among: potentially therapeutic molecules such as small molecules {e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drugs [e.g. taxol, oxaliplatine, etc.], etc.y peptides such as cytokines (e.g. TNF-a, I L-1 b, IL-6, etc.y

- proteins such as antibodies (e.g. Rituxan™, Herceptin™, Humira™, Benlysta™, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.y etc.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘A’ is different from said test agent ‘B’. In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘A’, said test agent ‘B’ and said test agent ‘C’ are different.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘A’ and said test agent ‘B’ are the same. In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘A’, said test agent ‘B’ and said test agent ‘C’ are the same.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘A’ and said test agent ‘B’ are the same, said test agent ‘A’ and said test agent ‘B’ being different from said test agent ‘O’. In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘B’ and said test agent ‘C’ are the same, said test agent ‘A’ and said test agent ‘B’ being different from said test agent ‘A’. In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said test agent ‘A’ and said test agent ‘B’ are the same, said test agent ‘A’ and said test agent ‘C’ being different from said test agent ‘B’.

In view of the above, it is understood that said first liquid, second liquid, third liquid and fourth liquid may comprise test agents. In other words, the nature of these liquids is not particularly important as they essentially serve as a vehicle (a carrier) for the test agents the diffusive gradients of which are to be generated in the cell (21 ; 210). These liquids must therefore be able to carry these test agents, i.e. they must be miscible with them. In addition, if microorganisms are requested when the microfluidic device of the invention is implemented, these liquids should be compatible with said microorganisms and non toxic, so as to allow their culture in an optimal manner.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, said microfluidic device comprising at least two assay chambers (20, 30, 40

- 20’, 30’, 40’) in the same row, placed on a (same) support (10), wherein said at least two assay chambers (20, 30, 40 - 20’, 30’, 40’) share at least one of said three independent channels (41a, 41d; 31a, 31d), in particular said at least two assay chambers (20, 30, 40 - 20’, 30’, 40’) share two of said three independent channels (41a, 41d - 28, 28’; 31a, 31d - 28, 28’) (see Figures 7, 8).

Concerning the references mentioned between the brackets, it has to be understood that the is used either to separate features belonging to one assay chamber from others or separate features belonging to one zone from others. For instance:

■ “at least two assay chambers (20, 30, 40 - 20’, 30’, 40’)” should be read as features 20, 30 and 40 belonging to the same assay chamber, the one which is on the left (see Figure 7), and features 20’, 30’ and 40’ belong to the same assay chamber, the one which is on the right (see Figure 7); and

■ “two of said three independent channels (41a, 41d - 28, 28’; 31a, 31d -28, 28’)” should be read as:

- features 41a, 41 d, 28 and 28’ belong to the same embodiment, the one presented Figure 7, features 41a and 41 d belong to the same zone (4), and features 28 and 28’ belong to the same zone (2); and

- features 31a, 31 d, 28 and 28’ belong to the same embodiment, the one presented Figure 8, features 31a and 31 d belong to the same zone (3), and features 28 and 28’ belong to the same zone (2).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, said microfluidic device comprising at least two assay chambers (20, 30, 40

- 20”, 30”, 40”) in the same column, placed on a (same) support (10), wherein said at least two assay chambers (20, 30, 40 - 20”, 30”, 40”) share at least one of said three independent channels (31a, 31d; 41a, 41d), in particular wherein said at least two assay chambers (20, 30, 40 - 20”, 30”, 40”) share two of said three independent channels (31a, 31 d - 28, 28”; 41a, 41 d - 28, 28”) (see Figures 9, 10).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, said microfluidic device comprising at least three assay chambers (20, 30, 40 - 20’, 30’, 40’ - 20”, 30”, 40”), placed on a (same) support (10), wherein: two of said at least three assay chambers (20, 30, 40 - 20’, 30’, 40’) are in the same row and share at least one of said three independent channels (41a, 41 d; 31a, 31 d), in particular said assay chambers (20, 30, 40 - 20’, 30’, 40’) in the same row share two of said three independent channels (41a, 41d - 28, 28’; 31a, 31d - 28, 28’); and two of said at least three assay chambers (20, 30, 40 - 20”, 30”, 40”) are in the same column and share at least one of said three independent channels (31a, 31 d; 41a, 41d), in particular said assay chambers (20, 30, 40 - 20”, 30”, 40”) in the same row share two of said three independent channels (31a, 31 d - 28, 28”; 41a, 41 d - 28, 28”) (see Figures 11, 12).

In this set-up, it is also understood that the implementation of the microfluidic device of the invention based on the Figure 11 involves that: a first liquid is injected in the network of interconnected channels (26, 27, 28, 29 - 26’, 27’, 28’, 29’ - 26”, 27”, 28”, 29”) via the inlet (25a), said first liquid diffusing in the cells (21 - 2T - 21”) via the contact point sets (x - x’ - x”); a second liquid is injected in the second channel (31a, 31 d) via the inlet (31b), said second liquid diffusing in the cells (21 - 21”) via the contact point sets (y - y”) by using the through-holes (24 - 24”) which makes the link between the cells (21 - 21”) located in zone (2) and the channel (31a, 31 d) located in zone (3); a third liquid is injected in the third channel (41a, 41 d) via the inlet (41b), said third liquid diffusing in the cells (21 - 2T) via the contact point sets (z - z’) by using the through- holes (32 - 32’) which makes the link between the through-holes (23 - 23’) located in zone (2) and the channel (41a, 41 d) located in zone (4) and the through-hole (23 - 23’) which makes the link between the cells (21 - 21’) located in zone (2) and the through- holes (32 - 32’) located in zone (3); a fourth liquid is injected in the fourth channel (31 ’a) via the inlet (31 ’b), said fourth liquid diffusing in the cell (2T) via the contact point set (y’) by using the through-hole (24’) which makes the link between the cell (21’) located in zone (2) and the channel (31 ’a) located in zone (3); and a fifth liquid is injected in the fifth channel (41”a) via the inlet (41”b), said fifth liquid diffusing in the cell (21”) via the contact point sets (z”) by using the through-holes (32”) which makes the link between the through-hole (23”) located in zone (2) and the channel (41”a) located in zone (4) and the through-hole (23”) which makes the link between the cell (21”) located in zone (2) and the through-hole (32”) located in zone (3); said first liquid can evacuate from the network of interconnected channels (26, 27, 28, 29 - 26’, 27’, 28’, 29’ - 26”, 27”, 28”, 29”) via the outlet (25b), said second liquid can evacuate from the second channel (31a, 31 d) via the outlet (31c), said third liquid can evacuate from the third channel (41a, 41 d) via the outlet (41c), said fourth liquid can evacuate from the fourth channel (31’a) via the outlet (31’c) and said fifth liquid can evacuate from the fifth channel (41”a) via the outlet (41 ”c). It should be also noted that injection of liquids into the microfluidic device of the invention can be made continuously {e.g. using an microfluidic automation tools). It should be also noted that if it is looked at the implementation of: the cell (21) as such: said first liquid, said second liquid and said third liquid respectively correspond to said aforementioned first liquid, said aforementioned second liquid that may comprise a test agent ‘A’ and said aforementioned third liquid that may comprise test agent ‘B’; the cell (2T) as such: o said first liquid and said third liquid respectively corresponds to said aforementioned first liquid and said aforementioned third liquid that may comprise test agent ‘B’; and o said fourth liquid corresponds to said aforementioned second liquid that may comprise a test agent ‘A’, and the cell (21”) as such: o said first liquid and said second liquid respectively corresponds to said aforementioned first liquid and said aforementioned second liquid that may comprise test agent ‘A’; and o said fifth liquid corresponds to said aforementioned third liquid that may comprise a test agent ‘B’. In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, wherein said at least three assay chambers (20, 30, 40 - 20’, 30’, 40’ - 20”, 30”, 40”) share one of said three independent channels (28, 28’, 28”) (see Figures 11, 12).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, said microfluidic device comprising at least m x n assay chambers, placed on a support (10), wherein: m is the number of assay chambers in each row and m is also the number of columns in the microfluidic device; and n is the number of assay chambers in each column and n is also the number of rows in the microfluidic device (see Figures 13, 14).

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, said microfluidic device comprising at least m x n assay chambers, placed on a support (10), wherein: m is a number chosen among: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, ,15, 16, 17, 18, 19 and 20; and n is a number chosen among: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, ,15, 16, 17, 18, 19 and 20.

In another embodiment, the subject matter of the invention concerns the microfluidic device as described above, said microfluidic device comprising at least m x n assay chambers, placed on a support (10), wherein m equals 4 and n equals 4, m equals 8 and n equals 8 or m equals 12 and n equals 12 (see Figures 13, 14).

Said m x n embodiments are the illustration of how the microfluidic device of the invention is multiplexed to create an array of compartmentalized photopatterned hydrogels or seedable porous scaffold, within which pairwise combinatorial chemical gradients of multiple test agents can be generated. Considering of the overall device, it is thus understood that only m + n +1 inlets are required to test combinations in m x n chambers. For example, in the 3 x 2 (m = 3 and n = 2) embodiments (/.e. 6 assay chambers), only 6 inlets are required to provide liquids to the 6 assay chambers:

- one located in the lower zone corresponding to the culture media;

- two located in the middle zone corresponding to the culture media comprising the test agent ‘AT and the culture media comprising the test agent ‘A2’; and - three located in the upper zone corresponding to the culture media comprising the test agent ‘BT, the culture media comprising the test agent ‘B2’ and the culture media comprising the test agent ‘B3’, in order to generate the following 6 combinations:

- A1/B1 , A1/B2 and A1/B3 diffusive gradients in the three assay chambers of the first column; and

- A2/B1 , A2/B2 and A2/B3 diffusive gradients in the three assay chambers of the second column.

In the particular case wherein m = n, it is thus understood that only 2m + 1 inlets (or 2n+1 inlets) are required to test combinations in m x n chambers. For example, in the 4 x 4 (m = 4 and n = 4) embodiments (/.e. 16 assay chambers, see Figure 13 top panel), only 9 inlets are required to provide liquids to the 16 assay chambers:

- one located in the lower zone corresponding to the culture media;

- four located in the middle zone corresponding to the culture media comprising the test agent ‘A1’, the culture media comprising the test agent ‘A2’, the culture media comprising the test agent ‘A3’ and the culture media comprising the test agent ‘A4’; and

- four located in the upper zone corresponding to the culture media comprising the test agent ‘BT, the culture media comprising the test agent ‘B2’, the culture media comprising the test agent ‘B3’ and the culture media comprising the test agent ‘B4’, in order to generate the following 16 combinations:

- A1/B1 , A1/B2, A1/B3 and A1/B4 diffusive gradients in the four assay chambers of the first column;

- A2/B1 , A2/B2, A2/B3 and A2/B4 diffusive gradients in the four assay chambers of the second column;

- A3/B1 , A3/B2, A3/B3 and A3/B4 diffusive gradients in the four assay chambers of the third column; and

- A4/B1 , A4/B2, A4/B3 and A4/B4 diffusive gradients in the four assay chambers of the fourth column.

In view of the above, it has to be pointed out that the microfluidic device of the invention comprises as many outlets as it comprises inlets since each channel comprises one inlet and one outlet. According to a second aspect, the subject matter of the invention concerns a process of manufacturing the microfluidic device as described above, said process: comprising a step of moulding the different zones followed by a step of assembling said zones; or being performed by 3D printing.

In a particular embodiment, the subject matter of the invention concerns the process of manufacturing the microfluidic device as described above, said process comprising a step of moulding the different zones followed by a step of assembling said zones (see Figure 16). The depicted process uses the sandwich mold fabrication process. The Si wafer is fabricated via standard photolithography process. A silanized transparency film is placed on top of the polymer precursor to locally inhibit its polymerization. Next, the fabricated zone is bonded to a substrate. Other zone(s) are fabricated and assembled similarly (see sections 1.3 and 1.4 of the example).

According to this particular embodiment, the subject matter of the invention concerns the process of manufacturing the microfluidic device as described above comprising n zones, said process comprising the following steps of: a. preparation of a Si wafer by modifying the surface of a wafer with Trichloro(1 H,1 H,2H,2H-perfluorooctyl)silane (PFOCTS); b. preparation of a functionalized transparent film by modifying the surface of a transparent film with [3-(2-Aminoethylamino)propyl]trimethoxysilane (AEAPTMS); c. addition of polydimethylsiloxane (PDMS) on said Si wafer; d. degassing said PDMS; e. laying said functionalized transparent film on top of said PDMS to get an assembly; f. covering said first assembly with foam pads, the whole assembly being then sandwiched with a custom-built metal clamp; g. baking at 90°C; h. releasing PDMS membrane from said Si wafer; i. laying said released PDMS membrane on a support or on a zone; j. plasma bonding to said PDMS membrane to said support or said zone; and k. releasing PDMS membrane from said functionalized transparent film, said steps a. to k. being repeated until the manufacturing of the zone n-1 and said process comprising for the manufacturing of the upper zone n (/.e. the last zone to made) the following steps of: a. preparation of a Si wafer by modifying the surface of a wafer with Trichloro(1 H,1 H,2H,2H-perfluorooctyl)silane (PFOCTS); b. addition of polydimethylsiloxane (PDMS) on said Si wafer; c. degassing said PDMS; d. baking at 90°C; e. releasing PDMS membrane from said Si wafer; f. laying said released PDMS membrane on zone n-1 ; and g. plasma bonding to said PDMS membrane to said zone n-1 .

In a particular embodiment, the subject matter of the invention concerns the process of manufacturing the microfluidic device as described above, said process being performed by 3D printing. In this case, a 3D model of the microfluidic device (containing all zones) is imported into a 3D printing software. The software breaks the 3D model into slices and take a bottom- up approach to sequentially print each slice. The type of 3D printer useful for such process may involve extrusion-based printing, such as fused deposition modelling (FDM) or Stereolithography (SLA) printing), knowing that the type of resin and technical parameters to use correspond to that recommended by the printer manufacturer.

According to a third aspect, the subject matter of the invention concerns an use of the microfluidic device as described above for generating at least one, in particular at least two, diffusive gradients by cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns a use of the microfluidic device as described above for generating at least three diffusive gradients by cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns the use as described above, wherein said diffusive gradient corresponds to a gradient of the test agent ‘A’, or a gradient of the test agent ‘B’, a gradient of the test agent ‘C’. In particular, the subject matter of the invention concerns the use as described above, wherein said diffusive gradient corresponds to a gradient of the test agent ‘A’. In particular, the subject matter of the invention concerns the use as described above, wherein said diffusive gradient corresponds to a gradient of the test agent ‘B’. In particular, the subject matter of the invention concerns the use as described above, wherein said diffusive gradient corresponds to a gradient of the test agent ‘C’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘A’ and the second diffusive gradient corresponds to a gradient of the test agent ‘B’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘B’ and the second diffusive gradient corresponds to a gradient of the test agent ‘A’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘A’ and the second diffusive gradient corresponds to a gradient of the test agent ‘C’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘C’ and the second diffusive gradient corresponds to a gradient of the test agent ‘A’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘B’ and the second diffusive gradient corresponds to a gradient of the test agent ‘C’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘C’ and the second diffusive gradient corresponds to a gradient of the test agent ‘B’.

In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘A’, the second diffusive gradient corresponds to a gradient of the test agent ‘B’ and the third diffusive gradient corresponds to a gradient of the test agent ‘C’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘C’, the second diffusive gradient corresponds to a gradient of the test agent ‘B’ and the third diffusive gradient corresponds to a gradient of the test agent ‘A’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘B’, the second diffusive gradient corresponds to a gradient of the test agent ‘C’ and the third diffusive gradient corresponds to a gradient of the test agent ‘A’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein the first diffusive gradient corresponds to a gradient of the test agent ‘B’, the second diffusive gradient corresponds to a gradient of the test agent ‘A’ and the third diffusive gradient corresponds to a gradient of the test agent ‘C’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘A’ is chosen among: potentially therapeutic molecules such as small molecules {e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.y peptides such as cytokines (e.g. TNF-a, I L-1 b, IL-6, etc.y

- proteins such as antibodies (e.g. Rituxan™, Herceptin™, Humira™, Benlysta™, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.y etc.

In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘B’ is chosen among: potentially therapeutic molecules such as small molecules (e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.y peptides such as cytokines (e.g. TNF-a, I L-1 b, IL-6, etc.y

- proteins such as antibodies (e.g. Rituxan™, Herceptin™, Humira™, Benlysta™, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.y etc.

In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘C’ is chosen among: potentially therapeutic molecules such as small molecules (e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.y peptides such as cytokines (e.g. TNF-a, I L-1 b, IL-6, etc.y

- proteins such as antibodies (e.g. Rituxan™, Herceptin™, Humira™, Benlysta™, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.y etc.

In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘A’ is different from said test agent ‘B’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘A’, said test agent ‘B’ and said test agent ‘C’ are different.

In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘A’ and said test agent ‘B’ are the same. In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘A’, said test agent ‘B’ and said test agent ‘C’ are the same.

In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘A’ and said test agent ‘B’ are the same, said test agent ‘A’ and said test agent ‘B’ being different from said test agent ‘C’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘B’ and said test agent ‘C’ are the same, said test agent ‘A’ and said test agent ‘B’ being different from said test agent ‘A’. In another embodiment, the subject matter of the invention concerns the use as described above, wherein said test agent ‘A’ and said test agent ‘B’ are the same, said test agent ‘A’ and said test agent ‘C’ being different from said test agent ‘B’.

In another embodiment, the subject matter of the invention concerns the use as described above, wherein the photocurable hydrogel scaffold (22; 220) or the seedable porous scaffold (22; 220) is adapted for the growth of microorganisms in a 3D environment, said photocurable hydrogel scaffold (22; 220) or said seedable porous scaffold (22; 220) comprising at least one microorganism chosen among eukaryotic cell and procaryotic cell.

In a particular embodiment, the subject matter of the invention concerns the use as described above, wherein said microorganism is an eucaryotic cell. In a particular embodiment, the subject matter of the invention concerns the use as described above, wherein said microorganism is a procaryotic cell.

According to a third aspect, the subject matter of the invention concerns a method for generating at least one, in particular at least two, diffusive gradients in the cell (21 ; 210) of the microfluidic device as described above, said cell (21 ; 210) comprising a polymerized photocurable hydrogel scaffold (22; 220) or a seedable porous scaffold (22; 220), said method comprising the following steps of: a. filling a first channel (e.g. 28; 301a) with a first liquid which flows from an inlet (e.g.

25a; 301 b) towards an outlet (e.g. 25b; 301c) and diffuses in said cell (21 ; 210); and b. filling a second channel {e.g. 31a; 250a) of said three independent channels with a second liquid comprising a test agent ‘A’ which flows from an inlet {e.g. 31 b; 250b) towards an outlet {e.g. 31c; 250c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘A’ inside said cell (21 ; 210).

According to this third aspect, the subject matter of the invention also concerns a method for generating at least one, in particular at least two, diffusive gradients in the cell (21 ; 210) of the microfluidic device as described above, said cell (21 ; 210) being pre-filled with a nonpolymerized photocurable hydrogel scaffold (22; 220), said method comprising the following steps of: a. polymerizing upon exposure to light said photocurable hydrogel scaffold (22; 220) in liquid phase so as to reticulate said photocurable hydrogel scaffold (22; 220) in a gel phase only inside said cell (21 ; 210) using a photomask comprising an optical window (11) as a guideline; b. eliminating from said first channel e.g. 28; 301a) the photocurable hydrogel scaffold (22; 220) in liquid phase not polymerized; c. filling said first channel {e.g. 28; 301a) with a first liquid which flows from an inlet {e.g. 25a; 301 b) towards an outlet {e.g. 25b; 301c) and diffuses in said cell (21 ; 210); and d. filling a second channel {e.g. 31a; 250a) of said three independent channels with a second liquid comprising a test agent ‘A’ which flows from an inlet {e.g. 31 b; 250b) towards an outlet {e.g. 31c; 250c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘A’ inside said cell (21 ; 210).

According to this third aspect, the subject matter of the invention also concerns a method for generating at least one, in particular at least two, diffusive gradients in the cell (21 ; 210) of the microfluidic device as described above, said method comprising the following steps of: a. filling said cell (21 ; 210) with a photocurable hydrogel scaffold (22; 220) in liquid phase using a first channel {e.g. 28; 301a) of said three independent channels; b. polymerizing upon exposure to light said photocurable hydrogel scaffold (22; 220) in liquid phase so as to reticulate said photocurable hydrogel scaffold (22; 220) in a gel phase only inside said cell (21 ; 210) using a photomask comprising an optical window (11) as a guideline; c. eliminating from said first channel {e.g. 28; 301a) the photocurable hydrogel scaffold (22; 220) in liquid phase not polymerized; d. filling said first channel (e.g. 28; 301a) with a first liquid which flows from an inlet (e.g. 25a; 301 b) towards an outlet (e.g. 25b; 301c) and diffuses in said cell (21 ; 210); and e. filling a second channel (e.g. 31a; 250a) of said three independent channels with a second liquid comprising a test agent ‘A’ which flows from an inlet (e.g. 31 b; 250b) towards an outlet (e.g. 31c; 250c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘A’ inside said cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns the method as described above, wherein the step b. (or a.) of polymerizing upon exposure to light said photocurable hydrogel scaffold (22; 220) is performed using 2-photon lithography, a laser beam, an UV light, a visible light or a near-IR light.

In another embodiment, the subject matter of the invention concerns the method as described above, wherein said UV I visible I near-IR light has a wavelength comprises from 365 nm to 800 nm, in particular from 400 nm to 450 nm, preferably of 405 nm.

In another embodiment, the subject matter of the invention concerns the method as described above, further comprising a step of filling a third channel (e.g. 41a; 260a) of said three independent channels with a third liquid comprising a test agent ‘B’ which flows from an inlet (e.g. 41 b; 260b) towards an outlet (e.g. 41c; 260c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘B’ inside said cell (21 ; 210). Consequently, it is understood that in a particular embodiment, the subject matter of the invention concerns the method as described above, said cell (21 ; 210) comprising a polymerized photocurable hydrogel scaffold (22; 220) or a seedable porous scaffold (22; 220), said method comprising the following steps of: a. filling a first channel (e.g. 28; 301a) with a first liquid which flows from an inlet (e.g. 25a; 301 b) towards an outlet (e.g. 25b; 301c) and diffuses in said cell (21 ; 210); b. filling a second channel (e.g. 31a; 250a) of said three independent channels with a second liquid comprising a test agent ‘A’ which flows from an inlet (e.g. 31 b; 250b) towards an outlet (e.g. 31c; 250c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘A’ inside said cell (21 ; 210); and c. filling a third channel (e.g. 41a; 260a) of said three independent channels with a third liquid comprising a test agent ‘B’ which flows from an inlet (e.g. 41 b; 260b) towards an outlet (e.g. 41c; 260c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘B’ inside said cell (21 ; 210). In another embodiment, the subject matter of the invention the method as described above, said cell (21 ; 210) being pre-filled with a non-polymerized photocurable hydrogel scaffold (22; 220), said method comprising the following steps of: a. polymerizing upon exposure to light said photocurable hydrogel scaffold (22; 220) in liquid phase so as to reticulate said photocurable hydrogel scaffold (22; 220) in a gel phase only inside said cell (21 ; 210) using a photomask comprising an optical window (11) as a guideline; b. eliminating from said first channel {e.g. 28; 301a) the photocurable hydrogel scaffold (22; 220) in liquid phase not polymerized; c. filling said first channel {e.g. 28; 301a) with a first liquid which flows from an inlet e.g. 25a; 301 b) towards an outlet {e.g. 25b; 301c) and diffuses in said cell (21 ; 210); d. filling a second channel {e.g. 31a; 250a) of said three independent channels with a second liquid comprising a test agent ‘A’ which flows from an inlet {e.g. 31 b; 250b) towards an outlet {e.g. 31c; 250c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘A’ inside said cell (21 ; 210); and e. filling a third channel {e.g. 41a; 260a) of said three independent channels with a third liquid comprising a test agent ‘B’ which flows from an inlet {e.g. 41b; 260b) towards an outlet {e.g. 41c; 260c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘B’ inside said cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns the method as described above, said method comprising the following steps of: a. filling said cell (21 ; 210) with a photocurable hydrogel scaffold (22; 220) in liquid phase using a first channel {e.g. 28; 301a) of said three independent channels; b. polymerizing upon exposure to light said photocurable hydrogel scaffold (22; 220) in liquid phase so as to reticulate said photocurable hydrogel scaffold (22; 220) in a gel phase only inside said cell (21 ; 210) using a photomask comprising an optical window (11) as a guideline; c. eliminating from said first channel {e.g. 28; 301a) the photocurable hydrogel scaffold (22; 220) in liquid phase not polymerized; d. filling said first channel {e.g. 28; 301a) with a first liquid which flows from an inlet {e.g. 25a; 301 b) towards an outlet {e.g. 25b; 301c) and diffuses in said cell (21 ; 210); e. filling a second channel (e.g. 31a; 250a) of said three independent channels with a second liquid comprising a test agent ‘A’ which flows from an inlet {e.g. 31 b; 250b) towards an outlet {e.g. 31c; 250c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘A’ inside said cell (21 ; 210); and f. filling a third channel e.g. 41a; 260a) of said three independent channels with a third liquid comprising a test agent ‘B’ which flows from an inlet {e.g. 41 b; 260b) towards an outlet {e.g. 41c; 260c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘B’ inside said cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns the method as described above, further comprising a step of filling a fourth channel {e.g. “j”) with a fourth liquid comprising a test agent ‘C’ which flows from an inlet towards an outlet and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘C’ inside said cell (21 ; 210). Consequently, it is understood that a particular embodiment, the subject matter of the invention concerns the method as described above, said cell (21 ; 210) comprising a polymerized photocurable hydrogel scaffold (22; 220) or a seedable porous scaffold (22; 220), said method comprising the following steps of: a. filling a first channel {e.g. 28; 301a) with a first liquid which flows from an inlet {e.g. 25a; 301 b) towards an outlet {e.g. 25b; 301c) and diffuses in said cell (21 ; 210); b. filling a second channel {e.g. 31a; 250a) of said three independent channels with a second liquid comprising a test agent ‘A’ which flows from an inlet {e.g. 31 b; 250b) towards an outlet {e.g. 31c; 250c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘A’ inside said cell (21 ; 210); c. filling a third channel {e.g. 41a; 260a) of said three independent channels with a third liquid comprising a test agent ‘B’ which flows from an inlet {e.g. 41 b; 260b) towards an outlet {e.g. 41c; 260c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘B’ inside said cell (21 ; 210); and d. filling a fourth channel {e.g. “j”) with a fourth liquid comprising a test agent ‘C’ which flows from an inlet towards an outlet and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘C’ inside said cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns the method as described above, said cell (21 ; 210) being pre-filled with a non-polymerized photocurable hydrogel scaffold (22; 220), said method comprising the following steps of: a. polymerizing upon exposure to light said photocurable hydrogel scaffold (22; 220) in liquid phase so as to reticulate said photocurable hydrogel scaffold (22; 220) in a gel phase only inside said cell (21 ; 210) using a photomask comprising an optical window (11) as a guideline; b. eliminating from said first channel {e.g. 28; 301a) the photocurable hydrogel scaffold (22; 220) in liquid phase not polymerized; c. filling said first channel {e.g. 28; 301a) with a first liquid which flows from an inlet e.g. 25a; 301 b) towards an outlet {e.g. 25b; 301c) and diffuses in said cell (21 ; 210); d. filling a second channel {e.g. 31a; 250a) of said three independent channels with a second liquid comprising a test agent ‘A’ which flows from an inlet {e.g. 31 b; 250b) towards an outlet {e.g. 31c; 250c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘A’ inside said cell (21 ; 210); e. filling a third channel {e.g. 41a; 260a) of said three independent channels with a third liquid comprising a test agent ‘B’ which flows from an inlet {e.g. 41b; 260b) towards an outlet {e.g. 41c; 260c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘B’ inside said cell (21 ; 210); and f. filling a fourth channel {e.g. “j”) with a fourth liquid comprising a test agent ‘C’ which flows from an inlet towards an outlet and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘C’ inside said cell (21 ; 210).

In another particular embodiment, the subject matter of the invention concerns the method as described above, said method comprising the following steps of: a. filling said cell (21 ; 210) with a photocurable hydrogel scaffold (22; 220) in liquid phase using a first channel {e.g. 28; 301a) of said three independent channels; b. polymerizing upon exposure to light said photocurable hydrogel scaffold (22; 220) in liquid phase so as to reticulate said photocurable hydrogel scaffold (22; 220) in a gel phase only inside said cell (21 ; 210) using a photomask comprising an optical window (11) as a guideline; c. eliminating from said first channel {e.g. 28; 301a) the photocurable hydrogel scaffold (22; 220) in liquid phase not polymerized; d. filling said first channel {e.g. 28; 301a) with a first liquid which flows from an inlet {e.g. 25a; 301 b) towards an outlet {e.g. 25b; 301c) and diffuses in said cell (21 ; 210); e. filling a second channel {e.g. 31a; 250a) of said three independent channels with a second liquid comprising a test agent ‘A’ which flows from an inlet {e.g. 31 b; 250b) towards an outlet (e.g. 31c; 250c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘A’ inside said cell (21 ; 210); f. filling a third channel {e.g. 41a; 260a) of said three independent channels with a third liquid comprising a test agent ‘B’ which flows from an inlet {e.g. 41 b; 260b) towards an outlet e.g. 41c; 260c) and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘B’ inside said cell (21 ; 210); and g. filling a fourth channel {e.g. “j”) with a fourth liquid comprising a test agent ‘C’ which flows from an inlet towards an outlet and diffuses in said cell (21 ; 210) thus forming a diffusive gradient of said test agent ‘C’ inside said cell (21 ; 210).

In another embodiment, the subject matter of the invention concerns the method as described above, wherein said photocurable hydrogel scaffold (22; 220) or said seedable porous scaffold (22; 220) is adapted for the growth of microorganisms in a 3D environment, said photocurable hydrogel scaffold (22; 220) or said seedable porous scaffold (22; 220) comprising at least one microorganism chosen among eukaryotic cell and procaryotic cell. In this set-up, it is understood that the method of the invention may further comprise a step of inoculation of said microorganism and/or a step of culturing said microorganism within the microfluidic device of the invention. It has to be pointed out that the seeding of said microorganism within the microfluidic device of the invention may be made at the same time as the photocurable hydrogel scaffold (22; 220) is cast into the microfluidic device of the invention insofar as the polymerization step involving light is not toxic to the microorganism.

In a particular embodiment, the subject matter of the invention concerns the method as described above, wherein said microorganism is an eucaryotic cell. In a particular embodiment, the subject matter of the invention concerns the method as described above, wherein said microorganism is a procaryotic cell.

In another embodiment, the subject matter of the invention concerns the method as described above, said liquids are culture media, said culture media being chosen among: Luria broth, Lennox broth, Mueller Hinton broth, M9, RPMI-1640 Medium, F12 Medium, Dulbecco's Modified Eagle Medium, etc. with or without additional components such as fetal bovine serum

In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘A’ is chosen among: potentially therapeutic molecules such as small molecules (e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.y peptides such as cytokines (e.g. TNF-a, I L-1 b, IL-6, etc.y

- proteins such as antibodies (e.g. Rituxan™, Herceptin™, Humira™, Benlysta™, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.y etc.

In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘B’ is chosen among: potentially therapeutic molecules such as small molecules (e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.y peptides such as cytokines (e.g. TNF-a, I L-1 b, IL-6, etc.y

- proteins such as antibodies (e.g. Rituxan™, Herceptin™, Humira™, Benlysta™, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.y etc.

In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘C’ is chosen among: potentially therapeutic molecules such as small molecules (e.g. antibiotics [e.g. gentamycin, chloramphenicol, ampicillin, etc.], antitumor drug [e.g. taxol, oxaliplatine, etc.], etc.y peptides such as cytokines (e.g. TNF-a, I L-1 b, IL-6, etc.y

- proteins such as antibodies (e.g. Rituxan™, Herceptin™, Humira™, Benlysta™, etc.) and growth factors (e.g. epidermal growth factor [EGF], growth hormone [somatotropin], platelet- derived growth factor [PDGF], etc.y etc.

In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘A’ is different from said test agent ‘B’. In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘A’, said test agent ‘B’ and said test agent ‘C’ are different. In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘A’ and said test agent ‘B’ are the same. In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘A’, said test agent ‘B’ and said test agent ‘C’ are the same.

In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘A’ and said test agent ‘B’ are the same, said test agent ‘A’ and said test agent ‘B’ being different from said test agent ‘C’. In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘B’ and said test agent ‘C’ are the same, said test agent ‘A’ and said test agent ‘B’ being different from said test agent ‘A’. In another embodiment, the subject matter of the invention concerns the method as described above, wherein said test agent ‘A’ and said test agent ‘B’ are the same, said test agent ‘A’ and said test agent ‘C’ being different from said test agent ‘B’.

In another embodiment, the subject matter of the invention concerns the method as described above, wherein the microfluidic device as described above comprises at least m x n assay chambers, placed on a support (10), wherein: m is the number of assay chambers in each row and m is also the number of columns in the microfluidic device; and n is the number of assay chambers in each column and n is also the number of rows in the microfluidic device, said method allowing to generate (or said method generating): in a given column a diffusive gradient of said test agent ‘A’ in each cell of said given column and in a given row a diffusive gradient of said test agent ‘B’ in each cell of said given row; or in a given column a diffusive gradient of said test agent ‘B’ in each cell of said given column and in a given row a diffusive gradient of said test agent ‘A’ in each cell of said given row, said test agent ‘A’ being liable to be different between at least two columns or between at least two rows and said test agent ‘B’ being liable to be different between at least two rows or between at least two columns.

In another embodiment, the subject matter of the invention concerns the method as described above, wherein said microfluidic device comprises at least m x n assay chambers, placed on a support (10), wherein: m is a number chosen among: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, ,15, 16, 17, 18, 19 and 20; and n is a number chosen among: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, ,15, 16, 17, 18, 19 and 20. In another embodiment, the subject matter of the invention concerns the method as described above, wherein said microfluidic device comprises at least m x n assay chambers, placed on a support (10), wherein m equals 4 and n equals 4, m equals 8 and n equals 8 or m equals 12 and n equals 12. 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. Furthermore, the present invention is illustrated by, but not limited to, the following Figures and Examples.

LIST OF FIGURES

Figure 1 : Schematic design of one possible embodiment of the microfluidic device of the invention with one assay chamber.

10: support; 11: optical window; 2: lower zone; 20: assay chamber; 21: cell; 22: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24: through-holes; 25a, 25b: fluidic inputs; 26-29: network of interconnected channels; x, y, z: contact point sets; 3: upper zone; 30: assay chamber; 31a: channel; 31b, 31c: fluidic inputs; 41a: channel; 41b, 41c: fluidic inputs and aa’: symmetric axis.

Figure 2: Schematic design of one possible embodiment of the microfluidic device of the invention with one assay chamber.

10: support; 11: optical window; 2: lower zone; 200: assay chamber; 210: cell; 220: photocurable hydrogel scaffold or seedable porous scaffold; 230: through-hole; 250a: channel; 250b, 250c: fluidic inputs; 260a: channel; 260b, 260c: fluidic inputs; x, y, z: contact point sets; 3: upper zone; 300: assay chamber; 310a: channel; 310b, 310c: fluidic inputs and aa’: symmetric axis.

Figure 3:

(A) Schematic design of one possible embodiment of the microfluidic device of the invention with one assay chamber.

10: support; 11: optical window; 2: lower zone; 20: assay chamber; 21: cell; 22: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24: through-holes; 25a, 25b: fluidic inputs; 26-29: network of interconnected channels; x, y, z: contact point sets; 3: middle zone; 30: assay chamber; 31a: channel; 31b, 31c: fluidic inputs; 32: through-hole; 4: upper zone; 40: assay chamber; 41a: channel; 41b, 41c: fluidic inputs, aa’: symmetric axis and AA: crosscutting axis of the lower panel.

(B) Schematic design of one possible embodiment of the microfluidic device of the invention with one assay chamber references but with size in micrometers (pm). The specified dimensions are provided to illustrate the microfluidic device of the invention without limiting the scope of the invention.

Figure 4: Schematic design of one possible embodiment of the microfluidic device of the invention with one assay chamber.

10: support; 11: optical window; 2: lower zone; 20: assay chamber; 21: cell; 22: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24: through-holes; 25a, 25b: fluidic inputs; 26-29: network of interconnected channels; x, y, z: contact point sets; 3: middle zone; 30: assay chamber; 31a: channel; 31b, 31c: fluidic inputs; 32: through-hole; 4: upper zone; 40: assay chamber; 41a: channel; 41b, 41c: fluidic inputs and aa’: symmetric axis.

Figure 5: Schematic design of one possible embodiment of the microfluidic device of the invention with one assay chamber.

10: support; 11: optical window; 2: lower zone; 20: assay chamber; 21: cell; 22: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24: through-holes; 25a, 25b: fluidic inputs; 26-29: network of interconnected channels; x, y, z: contact point sets; 3: middle zone; 30: assay chamber; 31a: channel; 31b, 31c: fluidic inputs; 32: through-hole; 4: upper zone; 40: assay chamber; 41a: channel; 41b, 41c: fluidic inputs and aa’: symmetric axis.

Figure 6: Schematic design of one possible embodiment of the microfluidic device of the invention with one assay chamber.

10: support; 11: optical window; 2: lower zone; 20: assay chamber; 21: cell; 22: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24: through-holes; 25a, 25b: fluidic inputs; 26-29: network of interconnected channels; x, y, z: contact point sets; 3: middle zone; 30: assay chamber; 31a: channel; 31b, 31c: fluidic inputs; 32: through-hole; 4: upper zone; 40: assay chamber; 41a: channel; 41b, 41c: fluidic inputs, aa’: symmetric axis and AA: crosscutting axis of the lower panel.

Figure 7: Schematic design of one possible embodiment of the microfluidic device of the invention with two assay chambers in the same row.

10: support; 11-11’: optical window; 2: lower zone; 20-20’: assay chamber; 21-21’: cell; 22-22’: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24-23’, 24’: through-holes; 25a, 25b: fluidic inputs; 26-29_26’-29’: network of interconnected channels; 3: middle zone; 30-30’: assay chamber; 31a-31’a: channel; 31b, 31c-31’b, 31 ’c: fluidic inputs; 32-32’: through- holes; 4: upper zone; 40-40’: assay chamber; 41a-41d: channel; 41b, 41c: fluidic inputs.

Figure 8: Schematic design of one possible embodiment of the microfluidic device of the invention with two assay chambers in the same row.

10: support; 11-11’: optical window; 2: lower zone; 20-20’: assay chamber; 21-21’: cell; 22-22’: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24-23’, 24’: through-holes; 25a, 25b: fluidic inputs; 26-29_26’-29’: network of interconnected channels; 3: middle zone; 30-30’: assay chamber; 31a-31d: channel; 31b, 31c: fluidic inputs; 32-32’: through-holes; 4: upper zone; 40-40’: assay chamber; 41a-41’a: channel; 41b, 41c-41’b, 41 ’c: fluidic inputs. Figure 9: Schematic design of one possible embodiment of the microfluidic device of the invention with two assay chambers in the same column.

10: support; 11-11”: optical window; 2: lower zone; 20-20”: assay chamber; 21-21”: cell; 22- 22”: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24-23”, 24”: through- holes; 25a, 25b: fluidic inputs; 26-29_26”-29”: network of interconnected channels; 3: middle zone; 30-30”: assay chamber; 31a-31d: channel; 31b, 31c: fluidic inputs; 32-32”: through- holes; 4: upper zone; 40-40”: assay chamber; 41a-41”a: channel; 41b, 41c-41”b, 41 ”c: fluidic inputs.

Figure 10: Schematic design of one possible embodiment of the microfluidic device of the invention with two assay chambers in the same column.

10: support; 11-11”: optical window; 2: lower zone; 20-20”: assay chamber; 21-21”: cell; 22- 22”: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24-23”, 24”: through- holes; 25a, 25b: fluidic inputs; 26-29_26”-29”: network of interconnected channels; 3: middle zone; 30-30”: assay chamber; 31a-31”a: channel; 31b, 31c-31”b, 31 ”c: fluidic inputs; 32-32”: through-holes; 4: upper zone; 40-40”: assay chamber; 41a-41d: channel; 41b, 41c: fluidic inputs.

Figure 11 : Schematic design of one possible embodiment of the microfluidic device of the invention with three assay chambers.

10: support; 11-11’-11”: optical window; 2: lower zone; 20-20’-20”: assay chamber; 21-21’- 21”: cell; 22-22’ -22”: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24-23’, 24’-23”, 24”: through-holes; 25a, 25b: fluidic inputs; 26-29_26’-29’_26”-29”: network of interconnected channels; 3: middle zone; 30-30’-30”: assay chamber; 31a, 31d-31’a: channel; 31b, 31c-31’b, 31’c: fluidic inputs; 32-32’ -32”: through-holes; 4: upper zone; 40-40’-40”: assay chamber; 41a, 41d-41”a: channel; 41b, 41c-41”b, 41 ”c: fluidic inputs.

Figure 12: Schematic design of one possible embodiment of the microfluidic device of the invention with three assay chambers.

10: support; 11-11’-11”: optical window; 2: lower zone; 20-20’-20”: assay chamber; 21-21’- 21”: cell; 22-22’ -22”: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24-23’, 24’-23”, 24”: through-holes; 25a, 25b: fluidic inputs; 26-29_26’-29’_26”-29”: network of interconnected channels; 3: middle zone; 30-30’-30”: assay chamber; 31a, 31d-31’’a: channel; 31b, 31c-31”b, 31 ”c: fluidic inputs; 32-32’ -32”: through-holes; 4: upper zone; 40-40’-40”: assay chamber; 41a, 41d-41’a: channel; 41b, 41c-41’b, 41 ’c: fluidic inputs. Figure 13: Schematic design of one possible embodiment of the microfluidic device of the invention with 4x4 or 8x8 assay chambers.

Figure 14: Schematic design of one possible embodiment of the microfluidic device of the invention with 12x12 assay chambers.

Figure 15: Schematic illustration of the working principles of the microdevice.

Figure 16: (a-c) Fabrication workflow, (d) 4x4 arrayed microdevice.

Figure 17: Numerical simulation of the concentration gradients of fluorescein (a) and sulforhodamine (b) as representatives of antibiotic molecules, (c), overlaid micrograph of the gradients of two antibiotics co-labeled with fluorescein and sulforhodamine, (d), lines of equally effective dosages (isoboles) in the simulated two-dimensional concentration space, (e), numerical simulation of glucose concentration in the chamber at different feeding rates with fresh MHB. The concentration remains considerably high to support the uniform growth of bacteria in the chamber.

Figure 18: Normalized growth of synergy evaluation of three antibiotic combinations in static conditions, a-c, Normalized growth of ATCC 25922 Escherichia coli after 18 h of incubation in presence of SLF/TRM, FOS/NIT, and AMP/CPR, respectively, d-f, Synergy score based on Bliss model for the aforementioned combinations.

Figure 19: Normalized growth of synergy evaluation of three antibiotic combinations in dynamic conditions, a-b, Normalized growth of ATCC 25922 Escherichia coli in presence of SLF/TRM after 12h (a) or 18h (b) of incubation, c-d, Normalized growth of ATCC 25922 Escherichia coli in presence of FOS/NIT after 12h (c) or 18h (d) of incubation, e-h, Synergy score based on Bliss model for the aforementioned combinations.

Figure 20: The growth patterns of E. coli 25922 in the presence of various concentrations of FOS and NIT. 1x MIC of FOS equivalent of 0.8 pg/ml and 1xMIC of NIT is equivalent of 5 pg/ml. The two antibiotics diffuse from either the left or the right side of the chamber and the fresh MHB flows on the top of the chamber. The white dashed lines indicate the approximate borders of the inhibition zones. The size of each panel is 1 mm x 1 mm.

Figure 21: The effect of NIT on the efficacy of FOS (a), and the effect of FOS on the efficacy of NIT (b). The bar size shows the area of confluency in the chamber. The indicated concentrations are the maximum concentrations in the antibiotic reservoirs, and thus, a linear dilution in each individual chamber would occur.

Figure 22: Schematic design of one possible embodiment of the microfluidic device of the invention with two zones and three assay chambers wherein at least the zone (2) is manufactured with a light blocking material.

10: support; 11-11’-11”: optical window; 2: lower zone; 20-20’-20”: assay chamber; 21-21’- 21”: cell; 22-22’ -22”: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24-23’, 24’-23”, 24”: through-holes; 25a, 25b: fluidic inputs; 26-29_26’-29’_26”-29”: network of interconnected channels; 3: middle zone; 30-30’-30”: assay chamber; 31a, 31d-31’a: channel; 31b, 31c-31’b, 31 ’c: fluidic inputs; 32-32’ -32”: through-holes; 4: upper zone; 40-40’-40”: assay chamber; 41a, 41d-41”a: channel; 41b, 41c-41”b, 41 ”c: fluidic inputs.

Figure 23: Schematic design of one possible embodiment of the microfluidic device of the invention with three zones and three assay chambers wherein at least the zone (2) is manufactured with a light blocking material.

10: support; 11-11’-11”: optical window; 2: lower zone; 20-20’-20”: assay chamber; 21-21’- 21”: cell; 22-22’ -22”: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24-23’, 24’-23”, 24”: through-holes; 25a, 25b: fluidic inputs; 26-29_26’-29’_26”-29”: network of interconnected channels; 3: middle zone; 30-30’-30”: assay chamber; 31a, 31d-31’a: channel; 31b, 31c-31’b, 31 ’c: fluidic inputs; 32-32’ -32”: through-holes; 4: upper zone; 40-40’-40”: assay chamber; 41a, 41d-41”a: channel; 41b, 41c-41”b, 41 ”c: fluidic inputs.

Figure 24: Schematic design of one possible embodiment of the microfluidic device of the invention with three zones and three assay chambers wherein only the zone (3) is manufactured with a light blocking material.

10: support; 11-11’-11”: optical window; 2: lower zone; 20-20’-20”: assay chamber; 21-21’- 21”: cell; 22-22’ -22”: photocurable hydrogel scaffold or seedable porous scaffold; 23, 24-23’, 24’-23”, 24”: through-holes; 25a, 25b: fluidic inputs; 26-29_26’-29’_26”-29”: network of interconnected channels; 3: middle zone; 30-30’-30”: assay chamber; 31a, 31d-31’a: channel; 31b, 31c-31’b, 31 ’c: fluidic inputs; 32-32’ -32”: through-holes; 4: upper zone; 40-40’-40”: assay chamber; 41a, 41d-41”a: channel; 41b, 41c-41”b, 41 ”c: fluidic inputs. EXAMPLES

1. Design and fabrication of the microfluidic platform

1.1 Design

The device has been designed in AutoCAD (Autodesk, student version). The conceptual illustration of an 8 x 8 arrayed device is shown in Figure 15. The design of one chamber (Figure 3) allows scale-up, thus by using the same concept, different array sizes are possible to achieve. Here a 4 x 4 arrayed design has been used. The device consists of a glass substrate and three microfabricated polymeric layers: The first layer contains all the microstructures for seeding and feeding the cells. The inner dimensions of one chamber are 1 mm x 1 mm x 40 pm (length, width, depth, respectively). All chambers and connecting microchannels will be filled with a photocurable hydrogel, embedded with bacterial cells. Illumination of UV light through a photomask, polymerizes the hydrogel inside the assay chambers (Figure 15b), while leaving the microchannels with uncured hydrogel precursor which will be washed in later steps. As shown in Figure 15c, on both sides of the chamber, two openings are embedded to allow the antibiotics to be in contact with the hydrogel. The top side of the chamber is in contact with the fresh culture medium (Mueller Hinton Broth, MHB) and the rest of the chamber is confined either by PDMS or glass, limiting the diffusion of the antibiotics to the desired directions. Upon loading the cell-hydrogel mixture, the hydrogel would fill the chambers (in the 1 ^st polymeric layer) and leak through the two openings on the sides of each chamber into the microchannels of the 2 ^nd and the 3 ^rd polymeric layers. By employing a photomask, the photo-sensitive polymer selectively crosslinks inside the chambers and the uncrosslinked polymer chains would wash out of the connecting microchannels on the first layer and the two topmost layers. Moreover, the geometry of the first layer is designed to avoid cross-contamination between the adjacent chambers by directing the flow to desired paths. The second and third layers carry the antibiotic solutions to the drug inlets that are on both sides of the hydrogel chambers. The first and second layers are membranes with through- holes that enable 3D fluidic communication between the three layers. The third layer is a thick PDMS with microfabricated features. Figure 15d shows how drug-drug interactions can change the growth pattern in the hydrogel.

1.2 Fabrication of the SU-8 masters

The first and second-layer master molds have been constructed by standard photolithography. Each mold has two layers of photoresist, a 40 pm layer to create the microfluidic channels and a 60 pm layer to create the through-holes in the membrane. The mold has been fabricated by spin-coating SU8-2035 photoresist at 2500 rpm for 30 s, followed by spin-coating SU8-2050 photoresist at 1750 rpm for 30 s. The master mold for the third layer has been fabricated by spin-coating SU8-2035 photoresist at 2500 rpm for 30 s. The master molds have been salinized by plasma treatment, followed by 20 minutes of exposure to PFOCTS vapor, and finally rinsing with HFE-7500.

1.3 Modification of the transparency film

To fabricate the through-holes membranes, it has needed to selectively inhibit PDMS polymerization. Therefore, transparency films have been functionalized with AEAPTMS to locally capture the Pt catalyst in the PDMS curing agent. When uncured PDMS comes into contact with the AEAPTMS-functionalized surface, lack of freely diffusing Pt ions prevents the polymerization reaction only on the surface of the transparency film (Karlsson JM et al. Fabrication and transfer of fragile 3D PDMS microstructures. Journal of Micromechanics and Microengineering. 2012 Aug;22(8).). Briefly, the transparency films have been immersed in 4% v/v AEAPTMS in a methanol anhydrous bath for 30 minutes. The films have been rinsed with copious amounts of MilliQ water. Finally, the films have been heated for 10 minutes at 110°C.

1.4 Soft lithography and assembly.

To fabricate the two membranes (the first and the second PDMS layers), the sandwich mold fabrication process has been used (Moraes C et al. Microfabricated platforms for mechanically dynamic cell culture. Journal of Visualized Experiments. 2010;(46).). Briefly, as depicted in Figure 16, each mold has been covered with 3 g of PDMS (10:1 base to curing agent), degassed it, and the activated transparency film has been placed on top of it. Then, the assembly has been covered with foam pads and the whole assembly has been sandwiched with a custom-built metal clamp. After baking at 90°C, the transparency film has been peeled off along with the PDMS membrane. Therefore, the PDMS membrane remained on the film. The carrier film facilitates the handing of the large (3” x 2”) flexible membrane. 30 g of PDMS have been casted on the third layer master mold using standard soft lithography procedures and have been baked it 70°C. The MJB4 mask aligner (Karl Suss) has been used to align the plasma-activated PDMS layers. 1.5 Numerical simulation

The concentration gradient pattern has been verified using ANSYS 2021 R2 (student version). The mesh had a total of 0.5 M elements. The simulation comprised of four domains: hydrogel (1 mm x 1 mm x 40 pm), sink (1.5 mm x 200 pm x 40 pm), compound A, and compound B (200 pm x 40 pm). The hydrogel has been modelled as a porous media, while the three latter domains have been modeled as fluid. For the porous media, the volume porosity has been assumed to be equal to 0.99 and the permeability equal to 2.5 x 10' ¹³ m ² . According to the water properties at 37°C, the fluid density has been considered equal to 993.3 kg/m ³ and dynamic viscosity has been considered equal to 0.6913 x 10' ³ Pa.s (Poon C. Measuring the density and viscosity of culture media for optimized computational fluid dynamics analysis of in vitro devices, doi.org/10.1101/2020.08.25.266221). Based on the diffusion properties of fluorescein and sulforhodamine, the diffusion coefficient of compound A has been set equal to 0.6 x 10' ⁵ cm ² /s (Galambos P, Forster FK. Micro Total Analysis Systems ’98. 1998.), and the diffusion coefficient of compound B has been set equal to 2.6 x 10' ⁶ cm ² /s (Evans SM et al.. A microfluidic method to measure small molecule diffusion in hydrogels. Materials Science and Engineering C. 2014 Feb 1 ;35(1):322-34.).

In addition, mass transport of glucose in the numerical domain has been investigated. The diffusion coefficient of glucose in water at 37 °C is 9 x 10' ⁶ cm ² /s (Cochran DM et al. Evolution of oxygen and glucose concentration profiles in a tissue-mimetic culture system of embryonic stem cells. Annals of Biomedical Engineering. 2006 Aug;34(8): 1247-58.). An uptake rate of 200 molecules/s.cell (Natarajan A, Srienc F. Dynamics of Glucose Uptake by Single Escherichia coli Cells) and a cell density of 0.20 OD in the hydrogel domain have been assumed. The cells consume uniformly within the hydrogel while the fresh media provides glucose from the top side of the hydrogel and also from the two openings for antibiotics. Transient simulations have been performed for 1800 seconds at MHB flow rates at 50, 5, and 0.5 pl/hr. 1.6 Chemicals and antibiotics

For surface modification of the wafer and the transparency film, Trichloro(1 H,1 H,2H,2H- perfluorooctyl)silane (PFOCTS, 448931 Sigma) and [3-(2- Aminoethylamino)propyl]trimethoxysilane (AEAPTMS, 440302 Sigma) have been respectively used. For dissolving AEAPTMS, Methanol anhydrous (322415 Sigma) has been used. To perform 3D culture, Gelatin methacryloyl (GelMA, 900496 Sigma-Aldrich) has been used as a scaffold and Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 900889 Sigma-Aldrich) has been used as a photoinitiator. To check the shape and stability of the concentration gradient in the microfluidic chip, Sulforhodamine 101 (S7635 Sigma) and Fluorescein isothiocyanate isomer I (F7250 Sigma) have been used. For bacterial culture, Cation-adjusted Mueller Hinton Broth 2 (CAMHB, 90922 Millipore) has been prepared according to the manufacturer's instructions. In the case of using Fosfomycin, the media has been supplemented with Glucose-6-phosphate (10127647001 Roche). Plate culture was performed in Mueller Hinton Agar (MHA, 70191 Millipore). Six antibiotics have been used: Nitrofurantoin (NIT, N7878 Sigma-Aldrich), Fosfomycin disodium salt (FOS, P5396 Sigma-Aldrich), Trimethoprim (TRM, 92131 Sigma-Aldrich), Sulfamonomethoxine (SLF, S0508 Sigma- Aldrich), Ampicillin (AMP, A9518 Sigma-Aldrich), and Ciprofloxacin (CPR, 17850 Sigma- Aldrich). Right before the experiments, 5.12 mg/ml NIT in Dimethyl sulfoxide (DMSO, D8418, Sigma), 25.6 mg/ml FOS in MilliQ water, 25.6 mg/ml TRM in DMSO, 5.12 mg/ml SLF in methanol, 25.6 mg/ml AMP in MilliQ water, and 4.8 mg/ml CPR in MilliQ water-1 N HCI (9:1) have been prepared.

1.7 Bacterial strain and medium

A master tube of ATCC 25922 Escherichia coli (ATCC, LGC Standards, France) has been purchased and E. coli has grew overnight according to the ATCC handling procedure. Tthe aliquots have been stored at -80°C in 25% glycerol. Then, streaked the bacteria on an MHA slant and cultured at 37°C overnight. On the day of the experiment, three to five isolated colonies of the same size from a fresh MHA plate have been transferred into 5 ml MHB and cultured for one to two hours. 1.8 Cell loading on platform and photo-patterning

100 mg of GelMA have been dissolved in 830 l of MHB at 50°C for 20 minutes and 70 pl of 25 mg/ml LAP have been added. The filter-sterilized solution was mixed in a 9:1 v/v ratio with concentrated bacterial to reach the desired cell density. The final ODeoo in this study ranged between 0.05 and 0.40. To load the cells, 5 pl of the above mixture have been injected from the inlet port of the L1 layer. A plastic photomask (Selba, Switzerland) has been aligned with the arrayed chambers and illuminated 375 nm UV light at 20 mW/cm ² for 10 seconds (Lighteningcure, Hamamatsu). Then, the unpolymerized hydrogel has been washed with MHB at 37°C.

1.9 Time-lapse fluorescence microscopy

To perform the experiments in microfluidics, an inverted fluorescence microscope (Zeiss Axio Observer 7) has been used, controlled with Zen 3.3 pro (Zeiss), and enclosed in a controlled temperature chamber to maintain the microfluidic chip and the culture medium reservoir at 37°C. A 10x EC Plan-Neofluar objective has been used for imaging and Hamamatsu Orca Flash 4 camera has been used to record the images. MHB has been pumped to the device at the constant flow rate of 5 pl/min by LineUp Flow EZ pressure controller (Fluigent, France), while injecting the antibiotic solutions at constant rate of 80pl/hr (Harvard Apparatus PHD 2000 pump). Images every 15 minutes for 16 hrs have been acquired. To verify the diffusion patterns and suitable functionality of the chip, the antibiotic solutions have been co-labeled with either Fluorescein or Sulforhodamine 101.

1.10 Antimicrobial susceptibility testing

The tests according to CLSI-M100-S28 [CLS] have been performed and established protocols (Wiegand I, et al. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols. 2008 Feb;3(2): 163-75.). Briefly, the broth microdilution method has been used in 96-well plates in triplicate in the range from 0 to 25.6 pg/ml by 2-fold dilutions increase for AMP, NIT, FOS, and TRM. For the two remaining antibiotics, the range of dilution was from 0 to 125 pg/ml for SLF and from 0 to 2 pg/ml for CPR. The results have been read according to CLSI and EUCAST instructions. 1.11 Checkerboard assay

For each pair of antibiotics, two checkerboard plates have been prepared and cultured in two ways. One in the static condition in a humidified incubator at 37°C, and the other in dynamic condition with continuous shaking at 37°C. In the latter, a humidity cassette has been used to minimize the evaporation from the plates. For dynamic cultures, the Tecan Spark microplate reader (Tecan Austria) has been used to incubate and measure the ODeoo every 15 minutes. For static cultures, the ODeoo has been measured after 18 hr of incubation. Independent experiments have been performed in duplicates.

1.12 Discovering pair interaction of antibiotics

Here the Bliss additivity model has been used to assess the interaction regime of two antibiotics. Based on this model, it has been assume that each drug in a combination behave independently of the other (Ocampo PS et al. Antagonism between bacteriostatic and bactericidal antibiotics is prevalent. Antimicrobial Agents and Chemotherapy. 2014;58(8):4573-82.; Cokol M et al. Systematic exploration of synergistic drug pairs. Molecular Systems Biology. 2011 ;7.). The reason for selecting this model to analyze the data is its suitability to measure the synergy of drug pairs with different mechanisms of action (Ocampo PS et al. Antagonism between bacteriostatic and bactericidal antibiotics is prevalent. Antimicrobial Agents and Chemotherapy. 2014;58(8):4573-82.). The python ‘synergy’ library has been used (Wooten DJ et al. A Python library for calculating, analyzing and visualizing drug combination synergy. Bioinformatics. 2021 May 15;37(10):1473-4.) to calculate the interaction.

2. Results

Figure 16d shows an optical micrograph of the device with 16 chambers, arranged in 4 rows and 4 columns. The multilayer design of the device allows the horizontal and vertical microchannels to pass over each other without crossing. Moreover, the through-holes membrane technology enables the delivery of antibiotic solutions to the intended chambers. Once an antibiotic solution flows through the second or third microfabricated layers, it reaches the hydrogel-filled chambers and starts to diffuse into the gel. On the other side of the hydrogel, the flow of fresh MHB without antibiotics creates a sink. The presence of sources and sink leads to a structured concentration gradient. The second antibiotic flows from the opposite side of the same chamber and diffuses into the hydrogel, creating a crossing concentration gradient. The gradients generated in each chamber rely only on the geometrical parameters. Therefore, after reaching the steady-state condition (in the order of 20 minutes for typical antibiotic molecule sizes), the concentration profile is independent of the size of the molecule or the hydrogel porosity.

Figures 17a-b shows the simulated concentration profiles of fluorescein and sulforhodamine at different MHB flow rates. At flow rates that are typical in microfluidics, MHB functions as an infinite sink. In addition, at a constant flow rate, the gradient structure reaches a stable state after 15 to 20 minutes (data not shown). The diffusion coefficients used in the simulations are measured in water. The true coefficient of diffusion of solutes in hydrogels depends on the volume fraction of the polymer, the radius of the solute, as well as the polymer-solvent condition (Amsden B. Solute diffusion within hydrogels. Mechanisms and models. Macromolecules. 1998 Nov 17;31 (23):8382-95.). However, the concentration profiles after reaching a steady-state remain independent of the coefficient of diffusion and the hydrogel parameters. Figure 17c shows the resulted concentration gradients of two fluorophores in one chamber of our microfluidic device, confirming the concept and simulation results. Figure 17d displays the combined effectiveness of the two compounds in the chamber. These lines can be used as a guideline to assess the interaction of the drugs. For example, if the expected inhibition zone in the absence of interaction between the two antibiotics is at 50%, then growth of bacterial colonies above this line (toward 100%) indicates antagonistic interaction between the selected pair of antibiotics. It has also been checked the availability of glucose in the hydrogel. The cells are encapsulated in a porous matrix that only allows the diffusion of molecules. While nutrients are consumed by the cells, fresh MHB always flows on top of the chamber, providing a nutrient-rich environment. In addition, the antibiotic solutions are diluted in MHB, creating a source of nutrients on the sides of each chamber. As shown in Figure 17e, the glucose concentration in reasonably uniform is the hydrogel at all flow rates, with a maximum of 20% reduction in the center of the hydrogel, the farthest point from the MHB sources.

Various antibiotics have been selected to reproduce all possible interactions between a given pair of antibiotics. Based on previous studies, FOS/NIT, SLF/TRM, and AMP/CPR create antagonistic, synergistic, and additive interactions, respectively. The interaction of the aforementioned combinations have been verified by the broth microdilution method. Figures 18a-c shows the normalized growth of the checkerboard assays cultured in a humidified static incubator. The interaction of those combinations using Bliss model have been evaluated (Figures 18d-f). Furthermore, it has been tested the identical checkerboard plates in dynamic culture in a microplate reader. Interestingly, the normalized growth (Figures 19a-d), and the pairwise interaction (Figures 19e-h) can significantly vary compared to the static culture. The variation in results, both in the absolute growth and synergy score, can be justified by the local shortage of nutrients and potentially a local decrease in antibiotic concentration at the lower of each well. The suppression of growth, arising from the two latter causes, does not uniformly change the growth and therefore, synergy score might differ in the two culture methods. Moreover, the interaction regime of antibiotics might be antagonistic in a certain concentration range and synergistic in another concentrations (Figure 19g). In the dynamic culture group, it has been also observed that the growth (Figure 19a) and the synergy score (Figure 19e) of a given pair of antibiotics after 12 hr of culture can match better with those of the static culture group (Figures 18a and 18d) after 18 hr. This suggests that in dynamic culture, where sufficient access to the nutrients and antibiotics, similar interactions happen but at a higher pace. Faster response time in the chip might originate from the dynamic environment and continuous flow of media.

To demonstrate the function of the chip, results of the FOS/NIT combination are shown in Figure 20. Based on the results of the checkerboard assay (Figures 18e, 19g and 19h), it has been expected to observe mainly antagonistic interactions between these two antibiotics. Elicited from the checkerboard assay, at elevated concentrations of FOS, regardless of NIT concentration, it has been expected to observe synergistic interaction (Figure 19g). The control chamber is shown in Figure 20a in which the cells are grown homogenously in the chamber in the absence of antibiotics. In Figures 20b-d, a gradual increase in the concentration of FOS results in an increased inhibition zone (shown in the dashed white line). The same pattern has been observed in Figures 20e, 20i, and 20m, in which the maximum concentration of NIT in the drug inlet on the right side of the chamber respectively increase. The rest of the panels in Figure 20 are exposed to both antibiotics. By comparing the reference chambers with one antibiotic (for example Figure 20c) with chambers with two antibiotics (for example Figure 20k), the interaction of the antibiotics can be identified. In this case, the presence of 4x MIC of NIT shrank the size of the FOS inhibition zone, which indicates the antagonistic effects of NIT on FOS. Interestingly, a comparison of the same chamber (Figure 20k) with the reference chamber for 8x MIC of NIT (Figure 20i), shows that the presence of FOS in the combination in the given concentration enlarges the inhibition zone. This finding reveals that the two antibiotics might have different effects on each other. In this case, the presence of 8x MIC of FOS in the chamber has synergistic effects on NIT, while the presence of 8x MIC of NIT has an antagonistic effect on the efficacy of FOS. This is an important feature of the proposed microdevice, which cannot be assessed in standard broth microdilution tests as in such an assay, the two antibiotics are well-mixed and the combined effect of them is measured.

The effect of the two antibiotics has been quantified in Figure 20. To measure the susceptibility of wild-type E. coli to each antibiotic, it has been calculated the area of the inhibition zones and compared it with the area of the negative control. Therefore, the individual effect of each antibiotic on the confluency of the bacteria is shown in Figure 21 . As expected, in the absence of either FOS or NIT, the confluency decreases in the chambers treated with higher dosages of antibiotics. However, adding NIT as the second antibiotic at 3.2 pg/ml and 6.4 pg/ml of FOS increases the confluency compared to the respective single-antibiotic confluency. An increment in the confluency compared to single-antibiotic confluency at the same concentration shows antagonistic interaction (for example the combination of 6.4 pg/ml of FOS with 40 pg/ml of NIT in Figure 21a). A reduction in confluency indicates synergistic interaction (for example the combination of 40 pg/ml of NIT with 6.4 pg/ml of FOS in Figure 21 b).