Microfluidic device for 3D cell and tissue culture

Abstract

The present invention relates to a microfluidic device (10) for 3D cell and tissue culture, wherein said microfluidic device (10) comprises at least one microfluidic unit (11) comprise a scaffold chamber (14) for housing a matrix (19) for culturing cells and / or acting as a barrier, in fluid communication with a media chamber in the form of a loop chamber (12) having a hollow section (16) for guiding a circular flow path of a fluid therein and a first capillary burst valve (15a) positioned between said scaffold chamber (14) and said loop chamber (12) arranged to selectively control the flow of fluid therebetween.

Description

[0001] MICROFLUIDIC DEVICE FOR 3D CELL AND TISSUE CULTURE

[0002] Technical Field of the Present Invention

[0003] The present invention relates to microfluidic devices and methods suitable for 3D cell and tissue culture to mimic spatial and functional interplays among cells and their microphysiological environment and determine cellular reactions.

[0004] Background of the Present Invention

[0005] Cells have the ability to respond to various types of environmental cues, and in many cases these cues induce directed cell migration towards or away from these signals. Cells can give chemotactic, haptotactic, and durotactic responses to chemical, biochemical, and physical gradients; and cell migration plays a critical role in development, angiogenesis, immune response, wound healing, and cancer metastasis. For example, breast cancer cells that express epidermal growth factor receptor move towards the source of epidermal growth factor source, and the unregulated chemotaxis of immune cells can lead to inflammatory diseases such as asthma and arthritis. Therefore, research in fields such as cancer, stem cells, immunology, development, endocrinology, and neuroscience require improved devices that can investigate interactions of cells with factors such as those mentioned above.

[0006] A number of recent studies have demonstrated that cells employ different migration mechanisms in 2D and 3D environments. Currently, most 2D and 3D gradient systems rely on diffusion-based mechanisms using large sink and source reservoirs. However, such gradients are not stable and cannot be used over long periods of time, which is especially important in 3D migration studies (Clark, Andrew G., et al. "3D cell migration in the presence of chemical gradients using microfluidics." Methods in Cell Biology. Vol. 147. Academic Press, 2018. 133-147). As a result, 3D cell culture models utilizing microfluidic systems have attracted attention because they can better mimic the in vivo microphysiological environment by incorporating various cell types into hydrogels, including native extracellular matrices (ECM), for long-term cell culture.

[0007] Microfluidics based systems comprise defined chambers and channels which provide temporal and spatial control such that gradients can be formed, and different cells can be positioned in a specific zone / zones. Therefore, by integrating 3D hydrogels into microfluidic chips, additional parameters of the microenvironment such as dynamic mechanical cues (e.g. fluid shear stress, interstitial fluid flow, etc.) or spatiotemporal chemical gradients (e.g. growth factor gradients) can be precisely controlled. These microfluidic cell culture systems can facilitate the formation of 3D microtissues with specific physiological functions that are now referred to as "organ-on-a-chip" models. This feature allows more realistic studies of cells in both health and disease states and improves drug testing approaches. Microfluidic systems are further advantageous in that they provide high-throughput analysis, low fabrication costs and portability. Due to the reduced dimensions of microfluidic chips, used material, such as cells and nutrient media, and waste volumes can be as low as picoliters. Using small volumes of unknown or toxic materials provides safe experimental study. It can also help reduce animal testing by providing an intermediate level of complexity between conventional 3D cell cultures and mouse models, allowing for more accurate active ingredient development as complexity increases.

[0008] Some examples in the art of microfluidic systems for 3D cell culture are available in WO 2012 / 050981, US 2023 / 147702, WO 2021 / 237195, US 2021 / 230527, US 11,066,630, US 10,227,556, WO 2021 / 215996, and EP 3

[0009] 932 555. However, there are certain disadvantages to these systems.

[0010] Current microphysiological systems mostly use closed chambers making it difficult to access the different zones either to load components or to retrieve them. Use of a syringe pump can improve reproducibility over manual loading; however, it is cumbersome to connect the syringe with each microfluidic chip via tubing, and bubbles may easily be generated when connecting and removing tubing, making it undesirable for high-throughput applications. In addition, gel loading with syringe pumps is unsuitable for certain hydrogels that require rapid mixing with a catalytic reagent to fully polymerize. Further, the flow requires use of pumps resulting in the same issues such as formation of cavities. And the fabrication process of current microphysiological systems require multiple steps and can therefore be problematic and costly.

[0011] Among others, a prior art publication in the technical field of the present invention attempting to ameliorate these issues may be referred to as US 2023 / 174909, which discloses a cell culture system for constructing a perfusable network of self-assembled cells comprising a multi-well plate embedded with microchannels connecting a central well with at least one inlet well and at least one outlet well, the central well for culturing seeded cells within an extracellular matrix, wherein the perfusable network allows perfusion through the microchannels connecting the central well with at least one inlet well and at least one outlet well.

[0012] Another reference can be given as WO 2021 / 206551 which discloses a microfluidic device comprising at least one cell culture unit for forming, culturing, growing and / or maintaining a 3D tissue structure. The cell culture unit comprises a culture chamber for culturing cells having a chamber outlet opening and a cell supply channel arranged to guide a microfluidic flow of liquid holding cells between a channel inlet and a channel outlet. The cell supply channel is provided with a flow inhibitor which is operable to selectively provide a flow inhibiting state or a flow permitting state depending on a fluid pressure at the flow inhibitor. The cell culture chamber further comprises at least two mutually spaced apart elastic support structures for elastically supporting a tissue formed in the culture chamber.

[0013] Another reference can be given as US 11,629,319 which discloses a microfluidic cell culture devices comprising perfusable vascular networks and a method of vascularizing a cell aggregate on said microfluidic device. The microfluidic device comprises one or more capillary pressure barriers allowing for formation of an extracellular matrix gel within a confined area of the network, in which cells can be cultured for different uses.

[0014] However, these systems and devices are not suitable for circular flow. Further, they mostly provide single gradients. Therefore, competitive response to multiple gradients is not possible to assess.

[0015] In order to address the problems in the prior art, the present invention proposes a microfluidic device for 3D cell and tissue culture where responses of cells to gradients and to other cells can be determined. Said microfluidic device comprises at least one microfluidic unit comprising a scaffold chamber for housing a matrix for culturing cells and / or acting as a barrier, in fluid communication with a media chamber in the form of a loop chamber having a hollow section for guiding a circular flow path of a fluid therein and a first capillary burst valve positioned between said scaffold chamber and said loop chamber arranged to selectively control the flow of fluid therebetween. The microfluidic device provides an open-top structure that can be manufactured in a single step and that can be easily loaded with fluid. The microfluidic device of the invention can be used as a 3D bioassay, for example for drug screening, personalized medicine, tissue engineering, wound healing, and other applications. The open-top structure allows 3D bioprinting to be directly realized into the microfluidic device.

[0016] Brief Description of the Technical Drawings

[0017] Accompanying drawings are given solely for the purpose of exemplifying a microfluidic device, whose advantages over prior art were outlined above and will be explained in brief hereinafter.

[0018] The drawings are not meant to delimit the scope of protection as identified in the Claims, nor should they be referred to alone in an effort to interpret the scope identified in said Claims without recourse to the technical disclosure in the description of the present invention.

[0019] Figure 1 demonstrates a top view of a concentric microfluidic unit of a microfluidic device according to the present invention.

[0020] Figure 2 demonstrates a perspective view of a concentric microfluidic unit of a microfluidic device according to the present invention.

[0021] Figure 3 demonstrates a top view of an eccentric microfluidic unit of a microfluidic device according to the present invention.

[0022] Figure 4 demonstrates a perspective view of an eccentric microfluidic unit of a microfluidic device according to the present invention.

[0023] Figure 5 demonstrates a top view of an alternative embodiment of a microfluidic unit having two scaffold chambers according to the present invention. Figure 6 demonstrates a top view of another alternative embodiment of a microfluidic unit having four scaffold chambers according to the present invention.

[0024] Figure 7 demonstrates a top view of an exemplary microfluidic device according to the present invention.

[0025] Figure 8 demonstrates a top view of another alternative embodiment of a microfluidic unit having at least one inclined section according to the present invention.

[0026] Figure 9 demonstrates a sectional view of a microfluidic unit of a microfluidic device along the line A-A' in Figure 8.

[0027] Detailed Description of the Present Invention

[0028] The present invention provides a microfluidic device (10) for 3D cell and tissue culture where responses of cells to gradients and to other cells can be determined. Said microfluidic device (10) is suitable for static and flow applications. Said microfluidic device (10) comprises at least one microfluidic unit (11) suitable for forming, culturing, growing and / or maintaining a 3D cell, tissue and / or barrier structure. Figures 1 and 2 illustrate an embodiment of said microfluidic unit (11), which comprises a scaffold chamber (14) for housing a matrix (19) for culturing cells and / or acting as a barrier, in fluid communication with a media chamber in the form of a loop chamber (12) having a hollow section (16) for guiding a circular flow path of a fluid therein and a first capillary burst valve (15a) positioned between said scaffold chamber (14) and said loop chamber (12) arranged to selectively control the flow of fluid therebetween. In one embodiment of the invention, said microfluidic unit (11) comprises at least one additional media chamber in fluid communication with said scaffold chamber (14) and a second capillary burst valve (15b) positioned between said scaffold chamber (14) and said media chamber arranged to selectively control the flow of fluid therebetween. Said media chamber may be in the form of a fluid chamber (13) of any shape as illustrated in the Figures, or in the form of another loop chamber (12).

[0029] In a preferred embodiment of the invention, said microfluidic unit (11) has an open-top structure, meaning that the top of the microfluidic unit (11) does not have a cover and fluids can be loaded therein without need for inlet / outlet structures. Fluids can be loaded into the chambers (14, 12, 13) of microfluidic unit (11) via the open-top by micropipetting or any other method known in the art. Suitable fluids include culture medium, physiological buffer solution, one or more biological molecule or chemical, cell laden matrix, cell free matrix or a combination thereof. This open-top structure that allows easy removal of cells and tissues with no detrimental effect on their structure and morphology. It is also more convenient to manufacture than closed top designs. However, in an alternative embodiment of the invention it is also possible for the scaffold chamber (14) to have a cover, having at least one inlet structure. In another alternative embodiment, said cover may have a second inlet for air flow.

[0030] The capillary burst valve designates a sudden expansion of a microchannel where the liquid meniscus is trapped at the beginning of the expansion. The liquid meniscus is stopped at the valve until the driving force overcomes the resisting capillary force, thus the flow regulation is easily achieved by controlling the driving force. When the valve bursts, the remaining channel is wetted by advancing liquid. The bursting pressure of the capillary burst valve is proportional to the surface tension of liquid and inversely proportional to the channel dimension. (Cho, Hansang, et al. "How the capillary burst microvalve works." Journal of colloid and interface science 306.2 (2007): 379- 385). Therefore, first capillary burst valve (15a) ensures that when matrix (19) fluid is loaded into said scaffold chamber (14) it is confined inside the scaffold chamber (14) without leaking into the adjacent media chambers and creates a first interface zone (18a) between the fluid inside the media chamber (12, 13) and the matrix (19) in the scaffold chamber (14) which is sufficient enough to ensure diffusion thereacross.

[0031] Matrix (19) loaded into said scaffold chamber (14) can consist of matrigel, collagen, laminin, agarose, polyacrylamide, biocompatible matrices, puramatrix, alginate, fibrin, hydrogels, or combinations thereof. Necessary environment is provided after the loading for polymerization in accordance with the type of the matrix (19), if necessary. For example, matrigel is loaded to the scaffold chamber (14) in a cold state, then the matrigel is ensured to polymerize by maintaining the microfluidic device (10) at 37 C. However, puramatrix polymerizes rapidly in salt solution, therefore its polymerization can be completed at the moment it is loaded to the scaffold chamber (14) when added to a salt solution. Matrix (19) can further comprise agents distributed uniformly or in controlled gradients in the matrix (19) or may be tethered to the matrix (19). Agents can be cells, cell-laden or cell-free matrix, polymer, hydrogels, culture medium, physiological buffer solution, cell conditioned culture medium, one or more biological or chemical molecules or a combination thereof. For example, agent may be a growth factor, or a pharmaceutical agent. Matrix (19) allows the diffusion of drugs and other small molecules that may interact with cells contained within the fluid-flow paths. Fluid loaded into media chambers (12, 13) include agents as described above. Matrix (19) and fluid can be loaded to their respective chambers concurrently or consecutively.

[0032] Cells for use in media chambers (12, 13) or for culturing in scaffold chamber (14) include cell lines, primary culture cells, biopsy cells, stem, endothelial, stromal, epithelial, immune, neuronal, connective, myocardial, hepato, renal, heart, liver, pancreas, muscle, brain, and kidney cells, and any kind of tumor cell or a combination thereof. For example, cancerous or normal cell lines can be used, solid or liquid biopsies obtained from patients can be used. Plant or microbial cells such as bacteria and yeast can also be used. After the cells settled down at the interface zone(s) (18a, 18b) the microfluidic device (10) can be incubated under suitable culture conditions. Cells used in media chambers (12, 13) and cells used as agents in scaffold chamber (14) can be the same or different.

[0033] Fluid flow within microfluidic device (10) is realized without the use of pumps by placing said microfluidic device (10) onto a rotator platform that does not require use of tubing. Rotator platforms offer distinct advantages over pump- driven systems including (i) lower medium volume requirements, which allows for the generation of appreciable concentrations of metabolites to observe real-time effects on other organs in the system; (ii) reduced cavitation-induced bubble formation generated by pumps that can block microfluidic flow; and (iii) increased scalability in those multiple systems (up to ~20 can be stacked on a single rocker within an incubator). (McAleer, Christopher W., et al. "Multi-organ system for the evaluation of efficacy and off-target toxicity of anticancer therapeutics." Science translational medicine 11.497 (2019): eaavl386.) The shape of scaffold chamber (14) can be oval, square, hexagonal or any multi-sided structure suitable for creating multiple gradients. The design of the scaffold chamber (14) is adaptable based whether microfluidic device (10) will be used under static and flow conditions and can comprise short or long side zones.

[0034] In one embodiment of the invention, said loop chamber (12) and said hollow section (16) are concentric, meaning that the microchannel formed within said loop chamber (12) has a uniform width. In an alternative embodiment, said loop chamber (127) and said hollow section (167) may be eccentric as shown in Figures 3 and 4. This creates varying fluid flowrates within said loop chamber (127) allowing the effects of biomechanical stresses, such as shear forces from fluid flow, to be observed. In the case pictured in the Figures, the fluid flowrate closest to the interface zone (18a7) will be greater than the fluid flowrate furthest from the interface zone (18a7).

[0035] In an alternative embodiment of the invention, said microfluidic unit (11) comprises a plurality of scaffold chambers (14). Figure 5 shows an exemplary embodiment wherein said microfluidic unit (ll777) comprises two scaffold chambers (14a777, 14c777) in fluid communication with a media chamber in the form of a loop chamber (12777) for guiding a circular flow path of a fluid therein and first capillary burst valves (15a777, 15c777) positioned between said scaffold chambers (14a777, 14c777) and said loop chamber (12777) arranged to selectively control the flow of fluid therebetween. Figure 6 shows another exemplary embodiment wherein said microfluidic unit (ll7777) comprises four scaffold chambers (14a7777, 14c7777, 14e7777, 14g7777) in fluid communication with a media chamber in the form of a loop chamber (127777) for guiding a circular flow path of a fluid therein and first capillary burst valves (15a7777, 15c7777, 15e7777, 15g7777) positioned between said scaffold chambers (14a7777, 14c7777, 14e"", 14g"") and said loop chamber (12"") arranged to selectively control the flow of fluid therebetween. Different types of matrix (19) and matrix (19) and agent combinations may be loaded into each scaffold chamber (14a, c"', 14a,c,e,g"") allowing for multiple tests to be performed simultaneously. These examples are not meant to be limiting and the skilled person can appreciate that said microfluidic unit (11) may comprise any number of scaffold chambers (14) and said scaffold chambers (14) may be in further fluid communication with additional media chambers (12, 13) as described above.

[0036] Microfluidic device (10) according to the present invention may comprise a plurality of microfluidic units (11) in an array, in particular in a two- dimensional array (Figure 7). Microfluidic device (10) is preferably in a multiarray format / multi-well format to be compatible with standard robotic plate readers and enable its use in in-vitro cell-based assays, pharmaceutical screening assays, toxicity assays, and the like. In one embodiment of the invention, microfluidic device (10) comprises at least one fluid reservoir (20) for storing fluids. In one embodiment of the invention, microfluidic units (11) are fluidly disconnected from each other. In another embodiment, of the invention microfluidic units (11) are in fluid connection with each other, much as via microchannels or other structures.

[0037] Preferably, the base (17) of said microfluidic unit (11) is configured to allow imaging of the interior of the scaffold chamber (14) through said base (17) using microscopy, including but not limited to in the visible spectrum. To that end at least part of the base (17) can be substantially transparent. Such imaging, for example video imaging, can advantageously enable visual studying and / or testing of the tissue structure in the scaffold chamber (14). In one embodiment of the invention, base (17) is configured to be flat. In an alternative embodiment of the invention, said base (17") structure contains at least one inclined section (21a" and 21b") that is inclined towards said scaffold chamber (14") in order to guide the fluid within media chambers (12", 13") towards the capillary burst valve(s) (15a", 15b") where interface zone(s) (18a", 18b") can be formed as discussed above (Figures 8 and 9). The skilled person will appreciate that the incline may be steeper or shallower than illustrated and the incline may have a sloping or curved structure.

[0038] Said microfluidic device (10) can be fabricated from a polymer selected from the group consisting of glass, a polymeric organosilicon compound, silicone, polydi methyl siloxane (PDMS), poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), polystyrene, and polycarbonate. Its simple design allows said microfluidic device (10) to be manufactured in one step by injection molding, without needing multiple steps such as manufacturing body and base separately and bonding them together.

[0039] The invention further includes a method for determining cell migration and invasion. There will be a gradient formation across the scaffold chamber (14) between loop chamber (12) and media chamber (13) such that cells in either loop chamber (12) or media chamber (13) can respond to the gradient. Chemoattractant agent can be present in either loop chamber (12) or media chamber (13). The response can exhibit itself as the migration of the certain type of cell, cell viability, expression of different genes, shape change etc.

[0040] Said method according to the present invention comprises the steps of: a) Loading cell-free or cell-laden matrix (19) to scaffold chamber (14) of microfluidic device (10), b) Loading cells to one loop chamber (12), c) Loading an agent to other media chamber (12 or 13), d) Incubating said microfluidic device (10) at appropriate cell culture conditions, e) Observing cell migration into the scaffold chamber (14).

[0041] Loading of the scaffold chamber (14) and media chambers (12 or 13), can also be carried out simultaneously. Loadings can be carried out into an empty microfluidic device (10) or a microfluidic device (10), previously loaded with culture medium or physiological buffer solution.

[0042] In an alternative embodiment of the method, before step b) a pre-loading step can be realized with endothelial cells, in order to create blood vessel interface mimic and observe extravasation of cells.

[0043] In an alternative embodiment of the method, in order to observe intravasation into the loop chamber (12), cell laden matrix (19) is used in step a), endothelial cells are loaded into the loop chamber (12) to mimic the blood vessel, cell migration from scaffold chamber (14) to loop chamber (12) is determined, for example, using microscopy.

[0044] In an alternative embodiment of the method, cell laden matrix (19) is used in step a), and hydrogels with different stiffness / mechanical properties are used in steps b) and c), in order to observe preference (attachment / invasion) of cells to different hydrogels.

[0045] In an alternative method, drug effects can be examined by comparing including but not limited to cell viability or vascularization or migration, in the presence and absence of drugs in one or more of the chambers (12, 13, 14).

[0046] In a nutshell, the present invention proposes a microfluidic device (10) for 3D cell and tissue culture, wherein said microfluidic device (10) comprises at least one microfluidic unit (11) comprising a scaffold chamber (14) for housing a matrix (19) for culturing cells and / or acting as a barrier, in fluid communication with a media chamber in the form of a loop chamber (12) having a hollow section (16) for guiding a circular flow path of a fluid therein and a first capillary burst valve (15a) positioned between said scaffold chamber (14) and said loop chamber (12) arranged to selectively control the flow of fluid therebetween.

[0047] In one variation of the present invention, said microfluidic unit (11) comprises at least one additional media chamber in fluid communication with said scaffold chamber (14) and a second capillary burst valve (15b) positioned between said scaffold chamber (14) and said media chamber arranged to selectively control the flow of fluid therebetween.

[0048] In a further variation of the present invention, said additional media chamber is a fluid chamber (13).

[0049] In a further variation of the present invention, said additional media chamber is a loop chamber (12).

[0050] In a further variation of the present invention, said loop chamber (12) and said hollow section (16) are concentric.

[0051] In a further variation of the present invention, said loop chamber (12) and said hollow section (16) are eccentric.

[0052] In a further variation of the present invention, said microfluidic unit (11"', 11"") comprises a plurality of scaffold chambers (14a, c"', 14a,c,e,g"") in fluid communication with a media chamber in the form of a loop chamber (12"', 12""). In a further variation of the present invention, said microfluidic device (10) comprises a base (17") structure that contains at least one inclined section (21a", 21b") that is configured to be inclined towards said scaffold chamber (14").

[0053] In a further variation of the present invention, said microfluidic device (10) comprises a plurality of microfluidic units (11) in an array.

[0054] In a further variation of the present invention, said matrix (19) is chosen from a group containing matrigel, collagen, laminin, agarose, polyacrylamide, biocompatible matrices, pura matrix, alginate, fibrin, hydrogels, and combinations thereof.

[0055] In a further variation of the present invention, said matrix (19) further comprises at least one agent distributed in or tethered to the matrix (19) uniformly or in gradient.

[0056] In a further variation of the present invention, said agent is chosen from a group containing cells, cell-laden or cell-free matrix, polymer, hydrogels, culture medium, physiological buffer solution, cell conditioned culture medium, one or more biological or chemical molecules, and a combination thereof.

[0057] The present invention further proposes a method for determining cell migration and invasion, wherein said method comprises the steps of: a) Loading cell-free or cell-laden matrix (19) to scaffold chamber (14) of microfluidic device (10) as described in any one of claims 2 to 12, b) Loading cells to one loop chamber (12), c) Loading an agent to other media chamber (12 or 13), d) Incubating said microfluidic device (10) at appropriate cell culture conditions, e) Observing cell migration into the scaffold chamber (14).

[0058] In a further variation of the present invention, said method further comprises a pre-loading with endothelial cells into loop chamber (12) step before step b), in order to create blood vessel interface mimic and observe extravasation of cells in the presence or absence of drugs in loop chamber (12) and / or media chamber (13) and / or scaffold chamber (14) under static or circular flow conditions.

[0059] In a further variation of the present invention, wherein cell-free or cell laden matrix (19) is used in step a), endothelial cells are loaded into the loop chamber (12) to mimic blood vessels, and endothelial cell migration from loop chamber (12) to scaffold chamber (14) is observed in the presence or absence of drugs in loop chamber (12) and / or media chamber (13) and / or scaffold chamber (14) under static or circular flow conditions.

[0060] In a further variation of the present invention, hydrogels with different biochemical and / or mechanical properties are used in step a) in order to observe preference of cells to different hydrogels in the presence or absence of drugs in loop chamber (12) and / or media chamber (13) and / or scaffold chamber (14) under static or circular flow conditions.

[0061] In a further variation of the present invention, said method comprises the steps of: a) Loading cell-laden matrix (19) to scaffold chamber (14) of microfluidic device (10) as described in any one of claims 2 to 12, b) Loading endothelial cells to one loop chamber (12) in order to create blood vessel interface mimic, c) Loading an agent to other media chamber (12 or 13), d) Incubating said microfluidic device (10) at appropriate cell culture conditions, e) Observing intravasation of cells from scaffold chamber (14) into loop chamber (14) in the presence or absence of drugs in loop (12) and / or media (13) and / or scaffold chambers under static or circular flow conditions.

[0062] The present invention further proposes a method for determining cell intravasation and extravasation, wherein said method comprises the steps of: a) Loading cell-laden matrix (19) to a scaffold chamber (14a'") of microfluidic device (10) as described in any one of claims 9 to 12, b) Loading cell-free or cell-laden matrix (19) to another scaffold chamber (14c'") of said microfluidic device (10), c) Loading endothelial cells to one loop chamber (12'") in order to create blood vessel interface mimic, d) Loading an agent to other media chamber (12a'" or 13a'"), e) Incubating said microfluidic device (10) at appropriate cell culture conditions, f) Observing intravasation of cells from scaffold chamber (14a'") into loop chamber (12'") in the presence or absence of drugs in loop chamber (12a'") and / or media chamber (13a'") and / or scaffold chamber (14a'") under static or circular flow conditions, g) Observing extravasation of cells from loop chamber (12) into another scaffold chamber (14c'") in the presence or absence of drugs in loop chamber (12a'") and / or media chamber (13a'") and / or scaffold chamber (14a'") under static or circular flow conditions. The present invention further proposes a method for creating circular flow wherein said microfluidic device (10) as described in any one of claims 2 to 12 is placed on a circular rotator and operating the rotator at desired rotations per minute with or without static intervals such that fluid in the loop chamber (12) flows in a circular manner, unidirectionally or bidirectionally.

[0063] In a further variation of the present invention, said method further comprises a pre-loading with endothelial cells into loop chamber (12) step before step b), and wherein during step d) said microfluidic device (10) is placed on a circular rotator and said rotator is operated at desired rotations per minute with or without static intervals, in order to create blood vessel interface mimic and observe effect of flow on endothelial cells in the presence or absence of drugs in loop chamber (12) and / or media chamber (13) and / or scaffold chamber (14) under circular flow conditions.

Claims

CLAIMS1) A microfluidic device (10) for 3D cell and tissue culture, wherein said microfluidic device (10) comprises at least one microfluidic unit (11) comprising a scaffold chamber (14) for housing a matrix (19) for culturing cells and / or acting as a barrier, in fluid communication with a media chamber in the form of a loop chamber (12) having a hollow section (16) for guiding a circular flow path of a fluid therein and a first capillary burst valve (15a) positioned between said scaffold chamber (14) and said loop chamber (12) arranged to selectively control the flow of fluid therebetween.2) A microfluidic device (10) as set forth in Claim 1, wherein said microfluidic unit (11) comprises at least one additional media chamber in fluid communication with said scaffold chamber (14) and a second capillary burst valve (15b) positioned between said scaffold chamber (14) and said media chamber arranged to selectively control the flow of fluid therebetween.3) A microfluidic device (10) as set forth in Claim 2, wherein said additional media chamber is a fluid chamber (13).4) A microfluidic device (10) as set forth in Claim 2, wherein said additional media chamber is a loop chamber (12).5) A microfluidic device (10) as set forth in any one of Claims 1 to 4, wherein said loop chamber (12) and said hollow section (16) are concentric.6) A microfluidic device (10) as set forth in any one of Claims 1 to 4, wherein said loop chamber (127) and said hollow section (167) are eccentric.7) A microfluidic device (10) as set forth in any preceding Claim, wherein said microfluidic unit (11"', 11"") comprises a plurality of scaffold chambers (14a, c"', 14a,c,e,g"") in fluid communication with a media chamber in theform of a loop chamber (12"', 12"").8) A microfluidic device (10) as set forth in any preceding Claim, wherein said microfluidic device (10) comprises a base (17") structure that contains at least one inclined section (21a", 21b") that is configured to be inclined towards said scaffold chamber (14").9) A microfluidic device (10) as set forth in any preceding Claim, wherein said microfluidic device (10) comprises a plurality of microfluidic units (11) in an array.10) A microfluidic device (10) as set forth in any preceding Claim, wherein said matrix (19) is chosen from a group containing matrigel, collagen, laminin, agarose, polyacrylamide, biocompatible matrices, puramatrix, alginate, fibrin, hydrogels, and combinations thereof.11) A microfluidic device (10) as set forth in any preceding Claim, wherein said matrix (19) further comprises at least one agent distributed in or tethered to the matrix (19) uniformly or in gradient.12) A microfluidic device (10) as set forth in Claim 11, wherein said agent is chosen from a group containing cells, cell-laden or cell-free matrix, polymer, hydrogels, culture medium, physiological buffer solution, cell conditioned culture medium, one or more biological or chemical molecules, and a combination thereof.13) A method for determining cell migration and invasion, wherein said method comprises the steps of: f) Loading cell-free or cell-laden matrix (19) to scaffold chamber (14) of microfluidic device (10) as described in any one of claims 2 to 12, g) Loading cells to one loop chamber (12),h) Loading an agent to other media chamber (12 or 13), i) Incubating said microfluidic device (10) at appropriate cell culture conditions, j) Observing cell migration into the scaffold chamber (14).14) A method as set forth in Claim 13, wherein said method further comprises a pre-loading with endothelial cells into loop chamber (12) step before step b), in order to create blood vessel interface mimic and observe extravasation of cells in the presence or absence of drugs in loop chamber (12) and / or media chamber (13) and / or scaffold chamber (14) under static or circular flow conditions.15) A method as set forth in Claim 13, wherein cell-free or cell laden matrix (19) is used in step a), endothelial cells are loaded into the loop chamber (12) to mimic blood vessels, and endothelial cell migration from loop chamber (12) to scaffold chamber (14) is observed in the presence or absence of drugs in loop chamber (12) and / or media chamber (13) and / or scaffold chamber (14) under static or circular flow conditions.16) A method as set forth in Claim 13, wherein hydrogels with different biochemical and / or mechanical properties are used in step a) in order to observe preference of cells to different hydrogels in the presence or absence of drugs in loop chamber (12) and / or media chamber (13) and / or scaffold chamber (14) under static or circular flow conditions.17) A method for determining cell intravasation, wherein said method comprises the steps of: f) Loading cell-laden matrix (19) to scaffold chamber (14) of microfluidic device (10) as described in any one of claims 2 to 12,g) Loading endothelial cells to one loop chamber (12) in order to create blood vessel interface mimic, h) Loading an agent to other media chamber (12 or 13), i) Incubating said microfluidic device (10) at appropriate cell culture conditions, j) Observing intravasation of cells from scaffold chamber (14) into loop chamber (14) in the presence or absence of drugs in loop (12) and / or media (13) and / or scaffold chambers under static or circular flow conditions.18) A method for determining cell intravasation and extravasation, wherein said method comprises the steps of: h) Loading cell-laden matrix (19) to a scaffold chamber (14a'") of microfluidic device (10) as described in any one of claims 9 to 12, i) Loading cell-free or cell-laden matrix (19) to another scaffold chamber (14c'") of said microfluidic device (10), j) Loading endothelial cells to one loop chamber (12'") in order to create blood vessel interface mimic, k) Loading an agent to other media chamber (12a'" or 13a'"), l) Incubating said microfluidic device (10) at appropriate cell culture conditions, m) Observing intravasation of cells from scaffold chamber (14a'") into loop chamber (12'") in the presence or absence of drugs in loop chamber (12a'") and / or media chamber (13a'") and / or scaffold chamber (14a'") under static or circular flow conditions, n) Observing extravasation of cells from loop chamber (12) into another scaffold chamber (14c'") in the presence or absence of drugs in loopchamber (12a'") and / or media chamber (13a'") and / or scaffold chamber (14a'") under static or circular flow conditions.19) A method for creating circular flow wherein said microfluidic device (10) as described in any one of claims 2 to 12 is placed on a circular rotator and operating the rotator at desired rotations per minute with or without static intervals such that fluid in the loop chamber (12) flows in a circular manner, unidirectionally or bidirectionally.20) A method as set forth in Claim 13, wherein said method further comprises a pre-loading with endothelial cells into loop chamber (12) step before step b), and wherein during step d) said microfluidic device (10) is placed on a circular rotator and said rotator is operated at desired rotations per minute with or without static intervals, in order to create blood vessel interface mimic and observe effect of flow on endothelial cells in the presence or absence of drugs in loop chamber (12) and / or media chamber (13) and / or scaffold chamber (14) under circular flow conditions.

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