A versatile microfluidic apparatus and an attachment thereof

Abstract

The present invention relates to a versatile microfluidic apparatus (100, 400) and a cell culture chamber attachment thereof. The present invention discloses a high-throughput and versatile multi-compartment organ-on-chip microfluidic apparatus to culture various 3D cell models and mimics the organ model with specific parenchyma and stroma. The cell culture chamber attachments (11) are replaceable and based on the variant of the 3D cell or tissue models. The apparatus (100, 400) is suitable for a variety of 3D cell models such as spheroid, biopsy tissue, organoid, hydrogel embedded tissue, 3D patterned, and 3D bio-printed culture models.

Description

A VERSATILE MICROFLUIDIC APPARATUS AND AN ATTACHMENT THEREOFFIELD OF THE INVENTION:

[0001] The present invention relates to the field of microfluidics. Particularly the present invention relates to a versatile microfluidic apparatus and an attachment thereof. More particularly, the present invention relates to the development of a high-throughput and versatile multi-compartment organ-on-chip model to culture various 3D cell models and mimic the organ model with specific parenchyma and stroma. Specifically, the present invention describes the use of multi-compartment organ-on-chip devices for drug efficacy and toxicity testing.BACKGROUND OF THE INVENTION:

[0002] The cornerstone of the drug research and screening processes is forecasting the effect of medications prior to human clinical trials. The expense of drug discovery is always increasing due to the poor predictability of 2D cell culture and animal models.

[0003] Pharmaceutical companies spend around 2B USD for new drug development. Against the huge drug discovery costs, the FDA approval rate remains only 10 % with the highest attrition rate for cancer drugs (96 %). The huge drug development cost is ultimately borne by the customers. Around 40 % of the drug compounds fail due to drug efficacy, and 30 % of them fail due to toxicity, which could not be captured by pre-clinical animal models. It is also possible to miss some of the promising candidates that did not show efficacy in animal models. Clearly, the animal pre-clinical models do not represent human pathophysiology completely. Moreover, drug metabolism in animal models differs from human drug metabolism.

[0004] As drug development is moving away from small molecule drugs to new modalities of drugs (cell therapies, gene therapies, and RNA therapeutics) human-relevant in vitro models will be more prevalent in pre-clinical drug discovery.

[0005] The convergence of microfabrication and tissue engineering led to the development of organ-on-a-chip technology, which provides an alternative to standard preclinical drug testing models. The potential of organ-on-a-chip technologies to imitate critical humanphysiological activities required for understanding medication effects improves preclinical safety and efficacy testing.

[0006] The organ-on-chips use primary organ-relevant cells and provide dynamic physiological conditions, faithfully imitating the human pathophysiology. The organ-on- chips carry the immense potential to enhance the success rate of preclinical testing and reduce drug development costs and time in clinical trials due to their predictive ability.

[0007] However, the reported organ-on-chips are low throughput, include complex handling protocols, require continuous membranes, and are non-versatile. Hence, much of the research work has been directed towards the development of alternative models of organ-on-chips platforms from all over the world.

[0008] Commercial organ-on-chip platforms are broadly classified into four types: a. Membrane based: Emulate, Dynamic42, Bi / ond, Beonchip, Netri, Alveolix, Akita, Biomimix, Chiron b. Capillary pinning / micro-compartmentalization / micro-patterning based: Mimetas, Aim Biotech, Vivodyne, Arcari biosciences, Beonchip, Netri, Qureator, Biomimx, Chiron, Synvivo, Xona microfluidics, Ananda c. Sacrificial / non- sacrificial material based: Numa, Voxcell bioinnovation, Organo Biotech d. Milliwells / microwells based: Hesperos, Miso chip, TissUse, CN Bio, Javelin biotech, React41ife, Kirkstall, Cherry Biotech, Revivo biosystems, Insphero, Quris Al

[0009] Many efforts have been made in the prior art to tackle the aforementioned concerns by providing various types of organ-on-chip platforms. Some of these prior arts are briefly discussed here:

[0010] US Patent 8357528 discloses a three-dimensional system comprising at least two layers, wherein each layer comprises channels divided longitudinally into two compartments by a centrally positioned membrane. Each compartment can comprise a different cell type (e.g., organ- specific cells in one compartment and a vascular supply in the second compartment). As a result, an aggregate of cell types can be contained in a single layer, which is integrated into a multi-layer system. The communication between compartments is controlled by porous or non-porous barriers.

[0011] Another US patent 10850274 discloses the microfluidic devices comprising patterned hydrogels with embedded cells or microtissues. The device provides the cellular composition compromising cells in polymerizable media and microfluidic device comprising an inlet channel, an outlet channel, a biocompatible cell culture chamber fluidly connected between the inlet and outlet and polymerizing the hydrogel with UV light to obtain cellular islands.

[0012] However, the existing platform technologies lack the versatility in terms of a single platform, which can culture various 3D models. Further, they require porous or non-porous continuous membranes.

[0013] Thus, there is a need for microfluidic platforms that can be used to culture all varieties of 3D cell culture models on a single platform, which reduces the complexity, eliminates the requirement for continuous membranes, and reduces the cost.OBJECTS OF THE INVENTION:

[0014] Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are listed herein below.

[0015] The primary object of the present invention is to provide a novel microfluidic platform, particularly in vitro organ-on-chips for providing micro-physiological environments.

[0016] Another object of the present invention is to develop a versatile microfluidic platform, an attachment thereof, and applications therefor.

[0017] Another object of the present invention is to provide a multi-compartment-based design model without the usage of continuous membranes particularly comprising two fluidic chambers that allow multi-cellular interactions between organ parenchyma and stroma to completely mimic various components of an organ.

[0018] Another object of the present invention is to develop an attachment-based design concept with an array of wells to achieve one or more cultures of spheroid, biopsy tissue, organoid, hydrogel embedded tissue, 3D patterned, and 3D bio-printed cell and / or tissue models; particularly to culture all the above 3D models on one platform.

[0019] These and other objects and advantages will become more apparent when reference is made to the following description and accompanying drawings.SUMMARY OF THE INVENTION

[0020] This summary is provided to introduce concepts related to a versatile microfluidic apparatus and a cell culture chamber attachment thereof. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0021] In an aspect of the present disclosure, a cell culture chamber attachment of a versatile microfluidic apparatus, for culturing cells and / or tissue is described. The cell culture attachment includes at least one array of cell culture chamber wells of a first geometry, symmetrically distributed on one surface of the cell culture chamber attachment. The first geometry of the cell culture chamber wells depends on a variety of 3D cell culture models. The diameter of each of the cell culture chamber wells of the at least one array of cell culture chamber wells is from 0.3 mm to 10 mm. The cell culture chamber attachment is of a third geometry to fit into at least one first cavity of a second geometry, of a cell culture chamber layer of the versatile micro fluidic apparatus.

[0022] In an embodiment of the present invention, a depth of each of the cell culture chamber wells of the at least one array of cell culture chamber wells is from 0.05 mm to 5.00 mm.

[0023] In another embodiment of the present invention, each of the cell culture chamber wells of the at least one array of cell culture chamber wells includes a plurality of vertical slits in a wall of the at least one array of cell culture chamber wells, and a width of the plurality of vertical slits is from 0.05 mm to 2.00 mm.

[0024] In another embodiment of the present invention, the variety of the 3D cell culture models is one of spheroid, biopsy tissue, organoid, hydrogel embedded tissue, 3D patterned, 3D co-culture, and 3D bio-printed culture models.

[0025] In another aspect of the present disclosure, a versatile microfluidic apparatus for culturing cells and / or tissue is described. The versatile microfluidic apparatus includes a base layer, a cell culture chamber layer, a polymeric layer, and a top fluidic chamber layer. The cell culture chamber layer includes at least one first cavity of a second geometry for placing at least one cell culture chamber attachment of a third geometry within the at least one first cavity. The at least one cell culture chamber attachment includes at least one arrayof cell culture chamber wells of a first geometry. The polymeric layer includes at least one array of holes. The top fluidic chamber layer includes at least one second cavity of a fourth geometry. The at least one second cavity of the top fluidic chamber layer corresponds to the at least one first cavity of the cell culture chamber layer. The cell culture chamber layer and the top fluidic chamber layer are separated by the polymeric layer in such a manner that the at least one array of cell culture chamber wells is fluidically connected to the second cavity of the top fluidic chamber layer through the at least one array of holes of the polymeric layer. The cell culture chamber layer includes a plurality of fluid channels of a fifth geometry that produces one or more desired flow rates through the plurality of fluid channels that simulate one or more micro-physiological environments or conditions of interest.

[0026] In an embodiment of the present invention, the top fluidic chamber layer and the cell culture chamber layer are controlled independently with crosstalk between them.

[0027] In another embodiment of the present invention, the top fluidic chamber layer and the cell culture chamber layer are isolated from each other.

[0028] In another embodiment of the present invention, the top fluidic chamber layer and the cell culture chamber layer communicate through hydrogels contained in the cell culture chamber attachment.

[0029] In another embodiment of the present invention, the at least one array of cell culture chamber wells of the cell culture chamber layer includes at least one cultured cell or tissue.

[0030] In another embodiment of the present invention, the at least one second cavity of the top fluidic chamber layer is configured for culturing cells on hydrogel contained in the cell culture chamber attachment.

[0031] In another embodiment of the present invention, the top fluidic chamber layer is cellular or acellular type.

[0032] In another embodiment of the present invention, the cellular-type top fluidic chamber layer is configured to culture organ-specific cells on top of the hydrogels contained in the cell culture chamber attachment.

[0033] In another embodiment of the present invention, the cell culture chamber layer is configured to culture parenchymal tissue in the cell culture chamber attachment, and the top fluidic layer is configured to culture stromal components on hydrogels.

[0034] In another embodiment of the present invention, the cell culture chamber layer is configured to culture stromal components in the cell culture chamber attachment, and the top fluidic layer is configured to culture parenchymal cells on hydrogels.

[0035] In another embodiment of the present invention, the thickness of the polymeric layer is from 1 pm to 2500 pm.

[0036] In another embodiment of the present invention, the microfluidic apparatus is made of one or more biocompatible materials.

[0037] In another embodiment of the present invention, the base layer and top fluidic chamber are configured to assemble the apparatus by at least one fastener.

[0038] In another embodiment of the present invention, the at least one fastener is hex nuts and socket head bolts.

[0039] In another embodiment of the present invention, the working volume of the microfluidic apparatus is between 0.1 ml and 1.0 ml.

[0040] In another embodiment of the present invention, a width of the plurality of fluid - channels of the fifth geometry is between 0.05 mm and 20 mm.

[0041] In another embodiment of the present invention, the cell culture chamber layer includes a plurality of inlets and outlets with the at least one first cavity of second geometry for placement of the cell culture chamber attachment of third geometry for organ-relevant cell culture.

[0042] In another embodiment of the present invention, the array of cell culture chamber wells in the cell culture chamber attachment is configured to support monolayer cell culture on top of them seeded in the top fluidic chamber or around them seeded in the plurality of fluid channels

[0043] In another embodiment of the present invention, the cell culture chamber attachment is configured to hold the cells / tissues to be cultured under dynamic flow for long-term studies.

[0044] In another embodiment of the present invention, a diameter of each of the cell culture chamber wells of the at least one array of cell culture chamber wells is from 0.3 to 10 mm.

[0045] In another embodiment of the present invention, a depth of each of the cell culture chamber wells of the at least one array of cell culture chamber wells is from 0.05 mm to5.00 mm.

[0046] In another aspect of the present disclosure, a versatile microfluidic apparatus for culturing cells and / or tissue is described. The versatile microfluidic apparatus includes a cell culture chamber layer, a polymeric layer, and a top fluidic chamber layer. The cell culture chamber layer includes a plurality of first cavities of a second geometry. Each of the plurality of first cavities is configured for placing a cell culture chamber attachment of a third geometry within their cavity. The at least cell culture chamber attachment includes a plurality of array of cell culture chamber wells of a first geometry. The polymeric layer includes a plurality of array of holes corresponding to the plurality of array of cell culture chamber wells of the at least cell culture chamber attachment. The top fluidic chamber layer includes a plurality of second cavities of a fourth geometry. The plurality of second cavities of the top fluidic chamber layer corresponds to the plurality of the first cavities of the cell culture chamber layer. The cell culture chamber layer and the top fluidic chamber layer are separated by the polymeric layer in such a manner that the plurality of array of cell culture chamber wells are fluidically connected to the plurality of second cavities of the top fluidic chamber layer through the plurality of array of holes of the polymeric layer. The cell culture chamber layer includes a plurality of fluid channels of a fifth geometry that produces one or more desired flow rates through the fluid channels that simulate one or more micro-physiological environments or conditions of interest.

[0047] In an embodiment of the present invention, the versatile microfluidic apparatus is fitted in a conventional 96-well plate or 384-well plate format.

[0048] Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.BRIEF DESCRIPTION OF DRAWINGS:

[0049] The illustrated embodiments of the subject matter will be understood by reference to the drawings, wherein like parts are designated by like numerals throughout. The following description is intended only by way of example and simply illustrates certain selected embodiments of devices, systems, and methods that areconsistent with the subject matter as claimed herein, wherein:

[0050] FIG. 1 illustrates a schematic representation of (a) a front view, (b) an isometric view, (c) a top view, of a non-assembled versatile microfluidic apparatus, and (d) and (e) an assembled view of a versatile microfluidic apparatus, in accordance with an exemplary embodiment of the present disclosure;

[0051] FIG. 2 illustrates a schematic representation of a cavity in a cell culture chamber layer where various cell culture chamber attachments or inserts can be placed for 3D cell culture in a microfluidic apparatus, in accordance with an exemplary embodiment of the present disclosure;

[0052] FIGs. 3a-3e illustrate schematic representations of various attachments or inserts for (a) biopsy tissue or spheroids culture, (b) acellular hydrogels / organoids / hydrogel embedded cell culture / cell-matrix interactions, (c) vascularization of tissues or hydrogel embedded spheroid culture, (d) multi-cellular interactions / cell patterning, and (e) 3D bioprinted tissue culture / 3D printed cell patterning, developed for culture of 3D cell culture in a versatile microfluidic apparatus or platform, in accordance with an exemplary embodiment of the present disclosure;

[0053] FIG. 4: illustrates a schematic representation of (a) a top view and isometric view of a versatile microfluidic apparatus in a high-throughput well plate format, and (b) an exploded assembly view of a versatile microfluidic apparatus in well plate format with a cell culture chamber layer and a top fluidic chamber layer separated by a polymeric layer, in accordance with an exemplary embodiment of the present disclosure;

[0054] FIG. 5 illustrates 3D MCF-7 tissue culture models stained with live (green) / dead (red) staining in respect of (a) 3D MCF 7 spheroids culture in a cell culture chamber attachment depicted in Fig. 3(a), (b) (from left-right) 3D MCF-7 culture inside hydrogel, 3D MCF-7 organoid tissue culture, MCF-7 interaction with collagen matrix in a cell culture chamber attachment depicted in Fig. 3(b), (c) 3D hydrogel embedded spheroid culture of MCF-7 in a cell culture chamber attachment depicted in Fig. 3(c), (d)cell patterning with hydrogel embedded culture of MCF-7 on right side and acellular hydrogel on left side in a cell culture chamber attachment depicted in Fig. 3(d), and (e) 3D bioprinted tissue culture in circular and rectangular shapes from hydrogel embedded MCF-7 cells in a cell culture chamber attachment depicted in Fig. 3(e), in a versatile microfluidicapparatus, in accordance with an exemplary embodiment of the present disclosure;

[0055] FIG. 6 illustrates a schematic representation of a versatile microfluidic apparatus with culturing of the organ-relevant cells (parenchyma / stroma) on top of hydrogels in a top fluidic chamber layer and culturing of the organ relevant cells (parenchyma / stroma) inside a cell culture chamber attachment, in accordance with an exemplary embodiment of the present disclosure; and

[0056] FIG. 7 illustrates a schematic representation of (a) an assembly of a versatile microfluidic device, (b) A549 seeded as a monolayer on top of acellular hydrogel, cultured in a cell culture chamber attachment depicted in Fig. 3b, in a top fluidic chamber layer (stained with live / dead staining), and (c) brightfield image showing A549 cells on top of acellular hydrogel, cultured in a cell culture chamber attachment depicted in Fig. 3b, in a top fluidic chamber layer, in accordance with an exemplary embodiment of the present disclosure.

[0057] The figures depict embodiments of the present subject matter for the purposes of illustration only. A person skilled in the art will easily recognize fromthe following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.DESCRIPTION OF THE INVENTION:

[0058] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0059] While the embodiments of the disclosure are subject to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the figures and will be described below. It should be understood, however, that it is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within thescope of the disclosure.

[0060] The terms “comprises”, “comprising”, or any other variations thereof used in the disclosure, are intended to cover a non-exclusive inclusion, such that a device, system, or assembly that comprises a list of components does not include only those components but may include other components not expressly listed or inherent to such system, or assembly, or device. In other words, one or more elements in a system or device proceeded by “comprises... a” does not, without more constraints, preclude the existence of other elements or additional elements in the system or device.

[0061] The present invention describes the development of an in vitro organ-on-chip versatile microfluidic platform / apparatus for providing in vivo micro-physiological conditions.

[0062] Researchers across the world use a variety of 3D cell culture models. However, conventional organ-on-chip platforms are not compatible with most such 3D models and lack versatility. Due to a lack of versatility, such platforms do not allow freedom for the researchers to implement their own human-relevant organ model. Further, these platforms often use continuous membranes.

[0063] To overcome the above-mentioned limitations, the inventors of the present application have developed a facile, high-throughput, and versatile multi-compartment organ-on-chip microfluidic platform or apparatus. Further, the platform allows the culture of all variants of 3D cell models such as spheroid, biopsy tissue, organoid, hydrogel embedded tissue, 3D patterned, and 3D bio-printed culture models. Moreover, multicellular interactions can be achieved through multi-compartment design. The present invention discloses a versatile microfluid platform with (i) multi-well attachments having at least one array of wells, and (ii) multi-compartment or multi-layer structure without the use of continuous membranes.

[0064] The above-mentioned technical features of the present invention provide technical advancement over the conventional organ-on-chip platforms. The multi-well attachment is very easy to work with for microfluidic and non-microfluidic users, as both of them are usually trained in using the conventional well-plates-based platform.

[0065] Thus, the present invention can culture all the aforementioned 3D models on a single platform. The multi-compartment and multi-well-based approach of the presentinvention is simpler for non-microfluidic users to implement, which is not the case with conventional platforms.

[0066] In one aspect, the present invention provides a single microfluidic platform with a cell culture chamber attachment having at least one array of wells to achieve one or more cultures of spheroid, biopsy tissue, organoid, hydrogel embedded tissue, 3D patterned, and 3D bio-printed cell or tissue models. The cultures of all the above 3D models are performed on a single microfluidic platform.

[0067] The present invention provides a multi-compartment or multi-layer structure without the usage of a continuous membrane, particularly having a cell culture chamber layer and a top fluidic chamber layer that allow multi-cellular interactions to completely mimic various components of an organ- specific such as organ parenchyma and stroma.

[0068] The present invention provides a mechanism that the cell culture chamber layer and the top fluidic chamber layer can be controlled independently with crosstalk between them.

[0069] In an embodiment, the cell culture chamber layer and the top fluidic chamber layer can remain isolated from each other.

[0070] In another embodiment of the present invention, the cell culture chamber layer and the top fluidic chamber layer communicate through hydrogels contained in the cell culture chamber attachments.

[0071] In one embodiment of the present invention, the cell culture chamber layer includes a cell culture chamber attachment that includes at least one array of wells for the 3D culturing of cells and / or tissues.

[0072] The top fluidic chamber layer includes cells or tissue that can be cultured on top of hydrogel contained in the cell culture chamber attachments. Further, the top fluidic chamber layer can either be cellular or acellular. Furthermore, if it is a cellular layer, it can culture organ-specific cells and / or tissues on top of hydrogels.

[0073] The cell culture chamber layer can culture parenchymal cells or tissues in the cell culture chamber attachments and the top fluidic chamber layer can culture stromal components on hydrogels or vice versa.

[0074] In yet another aspect, the present invention provides a mechanism to develop a multi-compartment or multi-layer structure by eliminating continuous porous membranesused in conventional platforms.

[0075] In an embodiment of the present invention, the diameter of at least one array of wells in the cell culture chamber attachments or inserts can vary from 0.3 mm - 10 mm. Further, the thickness of the polymeric layer can vary from 1-2500 pm.

[0076] The present invention also describes the use of biocompatible materials to fabricate the versatile microfluidic platform / apparatus and the cell culture chamber attachments.

[0077] Now, for better understanding, one or more embodiments of the present invention shall be described with respect to the earlier-mentioned drawings.

[0078] FIG. 1 illustrates a schematic representation of (a) a front view, (b) an isometric view, (c) a top view, of a non-assembled versatile microfluidic apparatus (100), and (d) and (e) an assembled view of a versatile microfluidic apparatus (100), in accordance with an exemplary embodiment of the present disclosure.

[0079] FIG. 2 illustrates a schematic representation of a cavity (7) in a cell culture chamber layer (3) where various cell culture chamber attachments / inserts can be placed for 3D cell culture in a microfluidic apparatus, in accordance with an exemplary embodiment of the present disclosure.

[0080] As illustrated, the versatile microfluidic apparatus (100) includes a top fluidic chamber layer (1), a polymeric layer (2), a cell culture chamber layer chamber (3), and a base layer (4).

[0081] The cell culture chamber layer (3) includes at least one first cavity (7) of a second geometry for placing at least one cell culture chamber attachment (11) of a third geometry within the at least one first cavity (7). The at least one cell culture chamber attachment (11) includes at least one array of cell culture chamber wells (12) (as shown at least in Fig. 3) of a first geometry. The cell culture chamber attachments (11) are placed in the first cavity (7) for 3D cell culture.

[0082] In an embodiment, the first geometry of the at least one array of cell culture chamber wells (12) includes a circular base (as shown at least in Fig. 3). However, the first geometry of the at least one array of cell culture chamber wells (12) may include another suitable base of another geometry, without deviating from the scope of the present disclosure.

[0083] In an embodiment, the second geometry of the at least one first cavity (7) and thethird geometry of the at least one cell culture chamber attachment (11) include a rectangular base (as shown at least in Fig. 2). However, the second geometry of the at least one first cavity (7) and the third geometry of the at least one cell culture chamber attachment (11) may include another suitable base of another geometry, without deviating from the scope of the present disclosure.

[0084] The top fluidic chamber layer (1) includes at least one second cavity (10) of a fourth geometry. The at least one second cavity (10) of the top fluidic chamber layer (1) corresponds to the at least one first cavity (7) of the cell culture chamber layer (3).

[0085] In an embodiment, the fourth geometry of the at least one second cavity (10) includes a rectangular base (as shown at least in Fig. 2). However, the fourth geometry of the at least one second cavity (10) may include another suitable base of another geometry, without deviating from the scope of the present disclosure.

[0086] The top fluidic chamber layer (1) and the cell culture chamber layer (3) are separated by the polymeric layer (2). The polymeric layer (2) includes at least one array of holes (8).

[0087] The cell culture chamber layer (3) and the top fluidic chamber layer (1) are separated by the polymeric layer (2) in such a manner that the at least one array of cell culture chamber wells is fluidically connected to the second cavity (10) of the top fluidic chamber layer (1) through the at least one array of holes (8) of the polymeric layer (2).

[0088] The cell culture chamber layer (3) includes a plurality of fluid channels (16) of a fifth geometry that produces one or more desired flow rates through them and simulates one or more micro-physiological environments or conditions of interest.

[0089] In an embodiment of the present invention, the fifth geometry of the plurality of fluid channels (16) includes a rectangular cross-section with a width varying between 0.05 mm and 20 mm. However, the fifth geometry of the plurality of fluid channels (16) may include another suitable geometry, without deviating from the scope of the present disclosure.

[0090] In an embodiment of the present invention, the top fluidic chamber layer (1) and the cell culture chamber layer (3) are controlled independently with crosstalk between them.

[0091] In another embodiment of the present invention, the top fluidic chamber layer (1)and the cell culture chamber layer (3) are isolated from each other.

[0092] In another embodiment of the present invention, the top fluidic chamber layer (1) and the cell culture chamber layer (3) communicate through hydrogels contained in the cell culture chamber attachment (11).

[0093] In another embodiment of the present invention, the at least one array of cell culture chamber wells (12) of the cell culture chamber layer (3) includes at least one cultured cell or tissue.

[0094] In another embodiment of the present invention, the at least one second cavity (10) of the top fluidic chamber layer (1) is configured for culturing cells on hydrogel contained in the cell culture chamber attachment (11).

[0095] In another embodiment of the present invention, the top fluidic chamber layer (1) is cellular or acellular type.

[0096] In another embodiment of the present invention, the cellular-type top fluidic chamber layer (1) is configured to culture organ- specific cells on top of the hydrogels contained in the cell culture chamber attachment (11).

[0097] In another embodiment of the present invention, the cell culture chamber layer (3) is configured to culture parenchymal tissue in the cell culture chamber attachment (11), and the top fluidic layer (1) is configured to culture stromal components on hydrogels.

[0098] In another embodiment of the present invention, the cell culture chamber layer (3) is configured to culture stromal components in the cell culture chamber attachment (11), and the top fluidic layer (1) is configured to culture parenchymal cells on hydrogels.

[0099] In another embodiment of the present invention, the thickness of the polymeric layer (2) is from 1 pm to 2500 pm.

[0100] In another embodiment of the present invention, the microfluidic apparatus (100) is made of one or more biocompatible materials.

[0101] In another embodiment of the present invention, the base layer (4) and the top fluidic chamber (1) are configured to assemble the apparatus (100) by at least one fastener.

[0102] In another embodiment of the present invention, the at least one fastener is hex nuts and socket head bolts.

[0103] The base layer (4) includes a plurality of holes for inserting hex nuts (5)and the top fluidic chamber layer includes corresponding holes for inserting socket head bolts (6). The versatile microfluidic apparatus (100) is assembled or sealed by the hex nuts (5) and the socket head bolts (6).

[0104] In another embodiment of the present invention, a working volume of the microfluidic apparatus (100) is between 0.1 ml and 1.0 ml.

[0105] In another embodiment of the present invention, a width of the fifth geometry of the plurality of fluid channels (16) is between 0.05 mm and 20 mm.

[0106] In another embodiment of the present invention, the cell culture chamber layer (3) includes a plurality of inlets (14) and a plurality of outlets (15) with the at least one first cavity (7) for placement of the cell culture chamber attachment (11) for organrelevant cell culture.

[0107] In another embodiment of the present invention, the at least one array of cell culture chamber wells (12) in the cell culture chamber attachment (11) is configured to support monolayer cell culture on top of them seeded in the top fluidic chamber layer (1) or around them seeded in the plurality of fluid channels (16).

[0108] In another embodiment of the present invention, the cell culture chamber attachment (11) is configured to hold the cells / tissues to be cultured under dynamic flow for long-term studies.

[0109] In another embodiment of the present invention, a diameter of each of the cell culture chamber wells of the at least one array of cell culture chamber wells (12) is from 0.3 to 10 mm.

[0110] In another embodiment of the present invention, a depth of each of the cell culture chamber wells of the at least one array of cell culture chamber wells (12) is from 0.05 mm to 5.00 mm.

[0111] In another embodiment of the present invention, the microfluidic apparatus (100) and the cell culture chamber attachment (11) are made of one or more biocompatible materials from, but not limited to, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass, polycarbonate (PC), polystyrene (PS), polyurethane (PU), cyclic olefin copolymer (COC), acrylonitrile butadiene styrene (ABS) polymer, polylactic acid (PLA), polyethylene terephthalate glycol (PETg), Polyethylene terephthalate (PET), Poly (methyl methacrylate) (PMMA), polyether ether ketone (PEEK), poly aryletherketone(PAEK), polytetrafluoroethylene (PTFE), polyurethane (PU), Nylon, polycarbonate (PC), polypropylene (PP), and Polytetrafluoroethylene (PTFE).

[0112] In another embodiment of the present invention, the cell culture chamber attachments (11) as well as the microfluidic apparatus (100) are made of other suitable materials such as, but not limited to, metallic materials, composites, or hydrogels.

[0113] In another embodiment of the present invention, the cell culture chamber attachments (11) as well as the microfluidic apparatus (100) are made of a suitable transparent material. However, other materials can also be used for designing and fabricating the cell culture chamber attachments (11) and / or the microfluidic apparatus (100), without deviating from the scope of the present disclosure.

[0114] In another embodiment of the present invention, the cell culture chamber layer (3) includes a plurality of inlets (14) and a plurality of outlets (15) with the first cavity (7) for placement of the cell culture chamber attachments (11) for organ-relevant cell culture (parenchyma / stroma).

[0115] In yet another aspect, the present invention provides a mechanism for preparing cells encapsulated in hydrogels, including biocompatible materials with > 50 % water content, and can be cross-linked with physical and chemical crosslinking.

[0116] The present invention is directed to develop a cell culture chamber attachment (11) with at least one array of cell culture chamber wells (12) to achieve one or more cultures of spheroid, biopsy tissue, organoid, hydrogel embedded tissue, 3D patterned, and 3D bio-printed cell and / or tissue models; particularly to culture all the above 3D models on a single versatile microfluidic platform or apparatus (100).

[0117] As illustrated in Fig. 2, the first cavity (7) of the cell culture chamber layer (3) is configured to place or receive the cell culture chamber attachments / inserts (11) suitably for various 3D cell cultures. The cell culture chamber attachments (11) hold the cells / tissues and culture them under dynamic flow for long-term studies.

[0118] FIGs. 3a-3e illustrate schematic representations of various attachments / inserts (11) in respect of (3a) biopsy tissue or spheroids culture, (3b) acellular hydrogels / organoids / hydrogel embedded cell culture / cell-matrix interactions, (3c) vascularization of tissues or hydrogel embedded spheroid culture, (3d) multi-cellular interactions / cell patterning, and (3e) 3D bio-printed tissue culture / 3D printed cellpatterning, developed for 3D cell culture in a versatile microfluidic apparatus / platform (100), in accordance with an exemplary embodiment of the present disclosure.

[0119] In an aspect, the cell culture chamber attachment (11) of a versatile microfluidic apparatus (100), for culturing cells and / or tissue, includes at least one array of cell culture chamber wells (12). The at least one array of cell culture chamber wells (12) is of a first geometry and is symmetrically distributed on one surface of the cell culture chamber attachment (11). The first geometry of the cell culture chamber wells (12) depends on a variety of 3D cell culture models. Each of the cell culture chamber wells of the at least one array of cell culture chamber wells (12) has a diameter range from 0.3 mm to 10 mm. The cell culture chamber attachment (11) is of a third geometry to fit into at least one first cavity (7) of a second geometry, of a cell culture chamber layer (3) of the versatile microfluidic apparatus (100).

[0120] In an embodiment of the present invention, each of the cell culture chamber wells of the at least one array of cell culture chamber wells (12) has a depth range from 0.05 mm to 5.00 mm.

[0121] In an embodiment of the present invention, each of the cell culture chamber wells of the at least one array of cell culture chamber wells (12) includes a plurality of vertical slits (13) in a wall of the at least one array of cell culture chamber wells (12). The plurality of vertical slits (13) has a width range from 0.05 mm to 2.00 mm. The plurality of vertical slits (13) work or function on the principle of meniscus pinning.

[0122] In an embodiment of the present invention, a structure of a cell culture chamber attachment (11) shown in Fig. 3(a) is for culturing 3D biopsy tissue or spheroids.

[0123] In an example embodiment, the structure of the cell culture chamber attachment (11) of Fig. 3(a) may be 7 mm in width and 22 mm in length. Each of the at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(a) may have an inner radius of 0.50 mm. The at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(a) may be protruded above the surface of the cell culture chamber attachment (11) by 0.50 mm. The thickness of the cell culture chamber attachment (11) of Fig. 3(a) may be 1 mm. The at least one array of cell culture chamber wells (12) of the cell culture chamber (11) of Fig.3(a) may have a depth, below the surface of the cell culture attachment (11), of 0.85 mm to trap the 3D spheroids or biopsy tissue. A width of each of the plurality of vertical slits (13) in the wall of the at least one array of cell culture chamber wells (12) may be 0.15 mm.

[0124] In another embodiment of the present invention, a structure of a cell culture chamber attachment (11) shown in Fig. 3(b) is for organoids / hydrogel embedded culture / cell-matrix interactions / acellular hydrogels.

[0125] In an example embodiment, each of the at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(b) may have an inner radius of 1.00 mm. The at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(b) may be protruded above the surface of the cell culture chamber attachment (11) by 1.00 mm. The thickness of the cell culture chamber attachment (11) of Fig. 3(b) may be 1 mm.

[0126] In another embodiment of the present invention, a structure of a cell culture chamber attachment (11) shown in Fig. 3(c) is for the vascularization of tissues and hydrogel-embedded spheroid culture.

[0127] In an example embodiment, each of the at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(c) may have an inner radius of 1.00 mm. The at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(c) may be protruded above the surface of the cell culture chamber attachment (11) by 1.00 mm. The thickness of the cell culture chamber attachment (11) of Fig. 3(c) may be 1mm. The at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(c) may have a depth, below the surface of the cell culture attachment (11), of 0.85 mm.

[0128] In another embodiment of the present invention, a structure of a cell culture chamber attachment (11) shown in Fig. 3(d) is for multi-cellular interactions and cell patterning.

[0129] In an example embodiment, each of the at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(d) may have an inner radius of 2.25 mm. The at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(d) may be protruded above the surface ofthe cell culture chamber attachment (11) by 1.00 mm.

[0130] As illustrated in Fig. 3(d), the at least one array of cell culture chamber wells (12) of the cell culture attachment (11) for 3D patterning further includes a rectangular divider (17) which works on the principle of meniscus pinning and helps to prevent hydrogel from leaking out to the other half of the well. Hence, one half of the circle can contain cell type 1 and the other half of the circle can contain cell type 2.

[0131] The rectangular divider (17) of the at least one array of cell culture chamber wells (12) of the cell culture attachment (11) of Fig 3d may have a width of 0.3 mm, a depth of 0.2 mm, and a length of 4.1 mm.

[0132] In another embodiment of the present invention, a structure of a cell culture chamber attachment (11) shown in Fig. 3(e) is for 3D bio-printed tissue culture / cell patterning with 3D printing.

[0133] In an example embodiment, each of the at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(e) may have an inner radius of 2.25 mm. The at least one array of cell culture chamber wells (12) of the cell culture chamber attachment (11) of Fig. 3(e) may be protruded above the surface of the cell culture chamber attachment (11) by 1.00 mm.

[0134] FIG. 4 illustrates a schematic representation of (a) a top view and isometric view of a versatile microfluidic apparatus (400) in well plate format, and (b) an exploded assembly view of a versatile microfluidic apparatus (400) in well plate format with a cell culture chamber layer (3) and a top fluidic chamber layer (1) separated by a polymeric layer (2), in accordance with an exemplary embodiment of the present disclosure.

[0135] As illustrated, the versatile microfluidic apparatus (400) for culturing cells and / or tissue includes a top fluidic chamber layer (1), a polymeric layer (2), and a cell culture chamber layer (3).

[0136] The assembly of the top fluidic layer (1), the polymeric layer (2), and the cell culture chamber layer (3) may include the usage of permanent bonding techniques including chemical glues, plasma activation, soldering, adhesive tapes, welding, corona discharge, or usage of other chemicals such as acetone, chloroforms, alcohol, or silanes and thermal techniques.

[0137] The cell culture chamber layer (3) includes a plurality of first cavities (7)of a second geometry. Each of the plurality of first cavities (7) is configured for placing a cell culture chamber attachment (11) of a third geometry within their cavity (7). The at least cell culture chamber attachment (11) includes a plurality of array of cell culture chamber wells (12) of a first geometry.

[0138] The polymeric layer (2) includes a plurality of array of holes (8) corresponding to the plurality of array of cell culture chamber wells (12) of the at least cell culture chamber attachment (11).

[0139] The top fluidic chamber layer (1) includes a plurality of second cavities (10) of a fourth geometry. The plurality of second cavities (10) of the top fluidic chamber layer (1) corresponds to the plurality of the first cavities (7) of the cell culture chamber layer (3).

[0140] The cell culture chamber layer (3) and the top fluidic chamber layer (1) are separated by the polymeric layer (2) in such a manner that the plurality of array of cell culture chamber wells (12) are fluidically connected to the plurality of second cavities (10) of the top fluidic chamber layer (1) through the plurality of array of holes (8) of the polymeric layer (2).

[0141] The cell culture chamber layer (3) includes a plurality of fluid channels (16) of a fifth geometry with a width between 0.05 mm and 20 mm that produces one or more desired flow rates through them and simulates one or more micro-physiological environments or conditions of interest.

[0142] In an embodiment of the present invention, the versatile microfluidic apparatus (400) is fitted in a 96-well plate or 384-well plate format.

[0143] In another embodiment of the present invention, the versatile microfluidic apparatus (400) includes a plastic well plate cover (9).

[0144] In one embodiment of the present invention, a single organ-on-chip versatile micro fluidic apparatus (100) of Figs. 1 and 2 can be fitted in a conventional wellplate format as shown in Fig. 4. This makes the microfluidic apparatus high-throughput by accommodating a plurality of the at least one cell culture chamber attachment (11) in their a plurality of corresponding first cavities (7). For example, there may be sixteen cell culture chamber attachments (11) in the sixteen first cavities (7) in one plate format.

[0145] In short, the design / structure of a versatile microfluidic apparatus / platformfor in vitro organ-on-chip cultures has been developed with various cell culture chamber attachments / inserts having at least one array of cell culture chamber wells that can be used for 3D culture of cells or tissues in high throughput mode.

[0146] Example 1 - Cell culture in a cell culture chamber attachment of a versatile microfluidic apparatus:

[0147] FIG. 5 illustrates 3D cell culture in developed attachments using MCF-7 cell line and live (green) / dead (red) staining (500) in respect of (a) 3D MCF 7 spheroids culture in a cell culture chamber attachment (11) depicted in Fig. 3(a), (b) (from left-right) 3D MCF-7 culture inside hydrogel, 3D MCF-7 organoid tissue culture, MCF-7 interaction with collagen matrix in a cell culture chamber attachment (11) depicted in Fig. 3(b), (c) 3D hydrogel embedded spheroid culture of MCF-7 in a cell culture chamber attachment (11) depicted in Fig. 3(c), (d) cell patterning with hydrogel embedded culture of MCF-7 on the right side and acellular hydrogel on the left side in a cell culture chamber attachment (11) depicted in Fig. 3(d), and (e) 3D bio-printed tissue culture in circular and rectangular shapes from hydrogel embedded MCF-7 cells in a cell culture chamber attachment (11) depicted in Fig. 3(e), in accordance with an exemplary embodiment of the present disclosure.

[0148] As illustrated, each 3D culture model has been validated with each of the at least one cell culture chamber attachment (11) placed in the microfluidic apparatus (100, 400). The cells or tissues utilized for the microfluidic platform (100, 400) are MCF 7 cells obtained from the National Centre for Cell Science (NCCS), Pune. The apparatus (100, 400) was conceptualized and fabricated using in-house additive manufacturing-based technology.

[0149] The cell viability staining shows that the green cells are live and red cells are dead cells represented in Figs. 5a-5e.

[0150] Fig. 5(a) depicts the culture of MCF 7 cells in a 3D spheroid format in the cell culture attachment (11) developed for biopsy tissue or spheroids (Fig. 3(a)) placed in the microfluidic apparatus (100, 400).

[0151] Fig. 5(b) depicts (from left-right) 3D MCF-7 culture inside hydrogel, 3D MCF-7 organoid tissue culture, MCF-7 interaction with collagen matrix in the cell culture chamber attachments (11) developed for acellular hydrogels, organoids, hydrogelembedded culture, and cell-matrix interactions (Fig. 3(b)) placed in the microfluidic apparatus (100, 400).

[0152] Fig. 5(c) depicts a 3D hydrogel embedded spheroid culture of MCF-7 in the cell culture chamber attachments (11) developed for vascularization of tissues or hydrogel-embedded spheroid culture (Fig. 3(c)) placed in the microfluidic apparatus (100, 400).

[0153] Fig. 5(d) depicts Cell patterning with hydrogel embedded culture of MCF- 7 on the right side and acellular hydrogel on the left side in the cell culture chamber attachments (11) developed for multicellular interactions / cell patterning (Fig. 3(d)) placed in the microfluidic apparatus (100, 400).

[0154] Fig. 5(e) depicts 3D bio-printed tissue culture in circular and rectangular shapes from hydrogel embedded MCF-7 cells in the cell culture chamber attachments (11) developed for a 3D bio-printed tissue culture / 3D printed cell patterning (Fig. 3(e)) placed in the microfluidic apparatus (100, 400).

[0155] The above-mentioned tissues were checked for viability on day 3. The cells have maintained high viability for a duration of 3 days of culture in each cell culture chamber attachment (11) placed in the microfluidic apparatus (100, 400).

[0156] Thus, 3D tissue culture models in the cell culture chamber attachments (11) placed in the microfluidic apparatus (100, 400) operate effectively. The cells remained highly viable in the apparatus (100, 400) for 3 days. Thus, these findings show that cells or tissue cultured on the cell culture chamber attachments (11) placed in the microfluidic apparatus (100, 400) can be used for at least 3 days according to the researcher's needs. However, the culture can also be propagated for more than 3 days.

[0157] Example 2 - Multi-cellular interaction with organ parenchyma and stroma (example used the attachment for hydrogel embedded culture as shown in Fig. 3b):

[0158] A cell culture chamber attachment for a hydrogel-embedded culture for multicellular interaction is depicted in Example 2 as shown in Fig. 6.

[0159] FIG. 6 illustrates a schematic representation (600) of a versatile microfluidic apparatus with culturing of the organ-relevant cells (parenchyma / stroma) on top of hydrogels in a top fluidic chamber layer and culturing of the organ relevant cells (parenchyma / stroma) inside a cell culture chamber attachment developed for hydrogelembedded cultures (Fig. 3(b)), in accordance with an exemplary embodiment of the present disclosure.

[0160] In an embodiment of the present invention, the organ-relevant cells (parenchyma / stroma) can be cultured on top of hydrogels in a top fluidic chamber layer (1) and organ-relevant cells (parenchyma / stroma) can be cultured inside a cell culture chamber attachment (11) in the cell culture chamber layer (3).

[0161] The above example and Fig. 6 depict that the cell culture chamber attachment (11) can culture organ-relevant cells inside hydrogels. Further, the top fluidic chamber layer (1) and the cell culture chamber layer (3) can be independently controlled with different fluidic medium compositions. At the same time, the top fluidic chamber layer (1) and the cell culture chamber layer (3) can interact with each other through the hydrogels contained in the cell culture chamber attachment (11).

[0162] Furthermore, the top fluidic chamber layer (1) can either contain cells or it can also remain acellular. Thus, the present invention can culture organ- specific parenchymal cells or tissues in isolation too.

[0163] Furthermore, if the top fluidic chamber layer (1) is cellular, it can culture organ- specific cells on top of hydrogels.

[0164] FIG. 7 illustrates a schematic representation (700) of (a) an assembly of a versatile microfluidic apparatus (100, 400) showing A549 cultured on hydrogel, particularly A549 lung epithelium cells and acellular hydrogel on the versatile microfluidic apparatus, (b) A549 seeded as a monolayer on top of acellular hydrogel in a top fluidic chamber layer (1) (stained with live / dead staining), and (c) brightfield image showing A549 cells on top of acellular hydrogel in a top fluidic chamber layer (1), in accordance with an exemplary embodiment of the present disclosure.

[0165] As illustrated in Fig.7, A549 lung epithelial cells were grown on top of the acellular hydrogel. Further, A549 cells were seeded as a monolayer in the top fluidic chamber layer (1) after acellular hydrogels were added to the cell culture chamber layer (3) in a cell culture chamber attachment (11) used for hydrogel embedded culture as depicted in Fig. 3(b).

[0166] Further, Fig. 7(a) depicts an assembly detail of a versatile microfluidic apparatus (100, 400) showing A549 cultured on hydrogel, particularly A549 lungepithelium hydrogel and acellular hydrogel on the versatile microfluidic apparatus (100, 400).

[0167] Further, Fig. 7(b) depicts A549 seeded as a monolayer on top of acellular hydrogel in a top fluidic chamber layer (1) of the versatile microfluidic apparatus (stained with live / dead staining).

[0168] Further, Fig. 7(c) depicts a brightfield image showing A549 cells on top of acellular hydrogel in the top fluidic chamber layer (1) of the versatile microfluidic apparatus (100, 400).

[0169] Thus, the multi-cellular interaction with organ-relevant cells particularly parenchyma or stroma can be cultured on top of hydrogels in the top fluidic chamber layer(1) and inside the cell culture chamber attachment (11) in the cell culture chamber layer (3). The example with A549 lung epithelial cells establishes the effective working of the microfluidic apparatus (100, 400) by culturing A549 cells on top of the acellular hydrogel.

[0170] EXAMPLE 3 depicts Table 1 for a comparison of the features of the present invention and some of the conventional organ-on-chip solutions / platforms taken from each category of classification introduced earlier.Table 1A few of the major advantages of the present invention over the conventional solutions: • The versatile micro fluidic apparatus / platform (100, 400) has better compatibility with 3D spheroids & biopsy tumours with respect to Hesperos, Emulate Bio, Mimetas, CN Bio, TissUse, Numa, and Aim Biotech.• The versatile microfluidic apparatus / platform (100, 400) has better compatibility with 3D bio-printed models compared to Hesperos, Emulate Bio, Mimetas, CN Bio, TissUse, Numa and Aim Biotech.• The versatile microfluidic apparatus / platform (100, 400) has medium to high relative throughput compared to Hesperos and Numa.• The versatile microfluidic apparatus / platform (100, 400) has better compatibility with hydrogel embedded cultures compared to Emulate Bio, CN Bio, and TissUse.• The versatile microfluidic apparatus / platform (100, 400) has better compatibility with immunohistochemistry compared to Emulate Bio, Mimetas, Numa, and Aim Biotech.

Claims

We claim:

1. A cell culture chamber attachment (11) of a versatile microfluidic apparatus (100, 400), for culturing cells and / or tissue, comprising: at least one array of cell culture chamber wells (12) of a first geometry, symmetrically distributed on one surface of the cell culture chamber attachment (11), wherein the first geometry of the cell culture chamber wells (12) depends on a variety of 3D cell culture models, wherein a diameter of each of the cell culture chamber wells of the at least one array of cell culture chamber wells (12) is from 0.3 mm to 10 mm, and wherein the cell culture chamber attachment (11) is of a third geometry to fit into at least one first cavity (7) of a second geometry, of a cell culture chamber layer (3) of the versatile microfluidic apparatus.

2. The cell culture chamber attachment (11) as claimed in claim 1, wherein a depth of each of the cell culture chamber wells (12) of the at least one array of cell culture chamber wells (12) is from 0.05 mm to 5.00 mm.

3. The cell culture chamber attachment (11) as claimed in claim 1, wherein each of the cell culture chamber wells of the at least one array of cell culture chamber wells (12) comprises a plurality of vertical slits (13) in a wall of the at least one array of cell culture chamber wells (12), and wherein a width of the plurality of vertical slits (13) is from 0.05 mm to 2.00 mm.

4. The cell culture chamber attachment (11) as claimed in claim 1, wherein the variety of the 3D cell culture models is one of spheroid, biopsy tissue, organoid, hydrogel embedded tissue, 3D patterned, 3D co-culture, and 3D bio-printed culture models.

5. A versatile microfluidic apparatus for culturing cells and / or tissue (100), comprising:• a base layer (4);• a cell culture chamber layer (3) comprising at least one first cavity (7) of a second geometry for placing at least one cell culture chamber attachment (11) of a third geometry within the at least one first cavity (7), the at least one cell culture chamber attachment (11) comprising at least one array of cell culture chamber wells (12) of a first geometry;• a polymeric layer (2) comprising at least one array of holes (8); and• a top fluidic chamber layer (1) comprising at least one second cavity (10) of a fourth geometry, the at least one second cavity (10) of the top fluidic chamber layer (1) corresponds to the at least one first cavity (7) of the cell culture chamber layer (3), wherein the cell culture chamber layer (3) and the top fluidic chamber layer (1) are separated by the polymeric layer (2) in such a manner that the at least one array of cell culture chamber wells (12) is fluidically connected to the second cavity (10) of the top fluidic chamber layer (1) through the at least one array of holes (8) of the polymeric layer (2), and wherein the cell culture chamber layer (3) comprises a plurality of fluid channels (16) of a fifth geometry that produces one or more desired flow rates through them and simulate one or more micro-physiological environments or conditions of interest.

6. The microfluidic apparatus (100) as claimed in claim 5, wherein the top fluidic chamber layer (1) and the cell culture chamber layer (3) are controlled independently with crosstalk between them.

7. The microfluidic apparatus (100) as claimed in claim 5, wherein the top fluidic chamber layer (1) and the cell culture chamber layer (3) are isolated from each other.

8. The microfluidic apparatus (100) as claimed in claim 5, wherein the top fluidic chamber layer (1) and the cell culture chamber layer (3) communicate through hydrogels contained in the cell culture chamber attachment (11).

9. The microfluidic apparatus (100) as claimed in claim 5, wherein the at least one array of cell culture chamber wells (12) of the cell culture chamber layer (3) comprises at least one cultured cell and / or tissue.

10. The microfluidic apparatus (100) as claimed in claim 5, wherein the at least one second cavity (10) of the top fluidic chamber layer (1) is configured for culturing cells on hydrogel contained in the cell culture chamber attachment (11).

11. The microfluidic apparatus (100) as claimed in claim 5, wherein the top fluidic chamber layer (1) is cellular or acellular type.

12. The microfluidic apparatus (100) as claimed in claim 5, wherein the cellular type top fluidic chamber layer (1) is configured to culture organ-specific cells on top of the hydrogels contained in the cell culture chamber attachment (11).

13. The microfluidic apparatus (100) as claimed in claim 5, wherein the cell culture chamber layer (3) is configured to culture parenchymal tissue in the cell culture chamber attachment (11), and the top fluidic layer (1) is configured to culture stromal components on hydrogels.

14. The microfluidic apparatus (100) as claimed in claim 5, wherein the cell culture chamber layer (3) is configured to culture stromal components in the cell culture chamber attachment (11), and the top fluidic layer (1) is configured to culture parenchymal cells on hydrogels.

15. The microfluidic apparatus (100) as claimed in claim 5, wherein the thickness of the polymeric layer (2) is from 1 pm to 2500 pm.

16. The microfluidic apparatus (100) as claimed in claim 5 is made of one or more biocompatible materials.

17. The microfluidic apparatus (100) as claimed in claim 5, wherein the base layer (4) and top fluidic chamber layer (1) are configured to assemble the apparatus by at least one fastener.

18. The microfluidic apparatus (100) as claimed in claim 17, wherein the at least one fastener is hex nuts (5) and socket head bolts (6).

19. The microfluidic apparatus (100) as claimed in claim 5, wherein a working volume of the microfluidic apparatus (100) is between 0.1 ml and 1.0 ml.

20. The microfluidic apparatus (100) as claimed in claim 19, wherein a width of the plurality of fluid channels (16) is from 0.05 mm to 20 mm.

21. The microfluidic apparatus (100) as claimed in claim 5, wherein the cell culture chamber layer (3) comprises a plurality of inlets (14) and outlets (15) with the at least one first cavity (7) for placement of the cell culture chamber attachment (11) for organ-relevant cell culture.

22. The microfluidic apparatus (100) as claimed in claim 5, wherein the at least one array of cell culture chamber wells (12) in the cell culture chamber attachment (11) is configured to support monolayer cell culture on top of them seeded in the top fluidic chamber (1) or around them seeded in the plurality of fluid channels (16).

23. The microfluidic apparatus (100) as claimed in claim 5, wherein the cell culture chamber attachment (11) is configured to hold the cells / tissues to be cultured under dynamic flow for long-term studies.

24. The microfluidic apparatus (100) as claimed in claim 5, wherein a diameter of each of the cell culture chamber wells of the at least one array of cell culture chamber wells (12) is from 0.3 to 10 mm.

25. The microfluidic apparatus (100) as claimed in claim 5, wherein a depth of each of the cell culture chamber wells of the at least one array of cell culture chamber wells (12) is from 0.05 mm to 5.00 mm.

26. A versatile microfluidic apparatus (400) for culturing cells and / or tissue, comprising:• a cell culture chamber layer (3) comprising a plurality of first cavities (7) of a second geometry, each of the plurality of first cavities (7) is configured for placing a cell culture chamber attachment (11) of a third geometry within their cavity, the at least cell culture chamber attachment (11) comprising a plurality of array of cell culture chamber wells (12) of a first geometry;• a polymeric layer (2) comprising a plurality of array of holes (8) corresponding to the plurality of array of cell culture chamber wells (12) of the at least cell culture chamber attachment (11); and• a top fluidic chamber layer (1) comprising a plurality of second cavities (10) of a fourth geometry, the plurality of second cavities (10) of the top fluidic chamber layer (1) corresponds to the plurality of the first cavities of the cell culture chamber layer (3), wherein the cell culture chamber layer (3) and the top fluidic chamber layer (1) are separated by the polymeric layer in such a manner that the plurality of array of cell culture chamber wells (12) are fluidically connected to the plurality of second cavities (10) of the top fluidic chamber layer (1) through the plurality of array of holes (8) of the polymeric layer (2), wherein the cell culture chamber layer (3) comprising a plurality of fluid channels of a fifth geometry (16) that produces one or more desired flow rates through the fluid channels that simulate one or more micro-physiological environments or conditions of interest.

27. The versatile microfluidic apparatus (400) as claimed in claim 26, is fitted in a 96-well plate or 384- well plate format (9).

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