Modular bioreactor device and kit thereof

Abstract

Bioreactor device comprising: a plurality of modular elements (1, 1', 1'', 2, 2', 2'', 3, 4, 5, 6, 7, 8, 9, 10), each of said modular elements (1, 1', 1'', 2, 2', 2'', 3, 4, 5, 6, 7, 8, 9, 10) comprising at least one well (14, 14', 14'', 14''', 15, 15', 15'', 15''', 16, 16', 16'', 17, 17', 18, 18') configured to house a biological culture; and a receptacle (100, 200) configured to house the plurality of modular elements (1, 1', 1'', 2, 2', 2'', 3, 4, 5, 6, 7, 8, 9, 10), the modular elements (1, 1', 1'', 2, 2', 2'', 3, 4, 5, 6, 7, 8, 9, 10) being configured to be reversibly placed inside the receptacle (100, 200) in such a way that the at least one well (14, 14', 14'', 14''', 15, 15', 15'', 15''', 16, 16', 16'', 17, 17', 18, 18') of one modular element (1, 1', 1'', 2, 2', 2'', 3, 4, 5, 6, 7, 8, 9, 10) is insulated from the at least one well (14, 14', 14'', 14''', 15, 15', 15'', 15''', 16, 16', 16'', 17, 17', 18, 18') of another modular element (1, 1', 1'', 2, 2', 2'', 3, 4, 5, 6, 7, 8, 9, 10).

Description

[0001]P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH "MODULAR BIOREACTOR DEVICE AND KIT THEREOF” DESCRIPTION TECHNICAL FIELD OF THE INVENTION The present invention relates to a modular multiwell bioreactor device for immunocompetent tissue culture. The bioreactor device comprises a receptacle designed to house a plurality of culture modular elements comprising interconnected wells. It forms subject matter of the present invention also a kit for use in a bioreactor comprising at least one modular element with interconnected wells and a receptacle configured to house the culture modules. STATE OF THE ART Reliable in vitro models are urgently needed to better understand key aspects of organ physiology and disease, opening new ways towards the development of innovative and effective treatments. Recent research trends have demonstrated important interest in establishing 3D engineered models of healthy and diseased tissues. Bioreactors have shown a pivotal role in maintaining cell viability and allowing tissue maturation in vitro. However, the absence of a proper immune system still determines a real challenge in defining robust and reliable cell and tissue models. This fact currently poses important limitations in the investigation of several biological processes, such as development and migration of malignancies and immune reactions derived from tissue remodelling processes. The lack of a representative immune system compartment is particularly important in the field of hepatology, due to the wide plethora of diseases involving immune cell participation (e.g., primary and secondary tumours, cancers, fibrosis, cirrhosis, etc.). On the other hand, the possibility to coculture in the same device tissue samples and immune cells derived from the same patient, leads to establishment of patient-specific models for personalised medicine. P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH In this context, several bioreactors were proposed in the attempt to support multiple tissue models during culture in vitro. However, the combination of these with cells from the immune system has not been fully investigated. More particularly, in the documents WO2022072421A1, WO2022072421A1, WO2006033935A2 and WO2022072421A1, “single-block” multiwell plate-like devices were described. The disclosed devices are represented by a single multiwell plate with different well configurations and interconnections, among which: fibrous hydrogels (WO2022072421A1), total well submersion with common culture medium (WO2022072421A1), small canals present at the bottom of each well (WO2006033935A2) and partial interconnections (WO2022072421A1). However, all the cited bioreactors are “single-block” devices and are characterized by a fixed design, not customizable to the different biological tissues obtained from biopsies and / or surgeries that are often quite irregular and usually depend on the type of resection. If it is not possible to customize the design to the specific tissue, it often happens that several wells can remain unused, with a consequent increase of the plastic waste. Moreover, despite the possibility of multiple interconnections between wells, in all cited devices the well connections are intrinsically embedded in the bioreactor and, thus, they do not allow for actively modifying the inter-well connections during the use of the device itself. Instead, a key aspect in modeling the immune system together with tissues, is to modify the connections during use to allow the immune cells to temporarily circulate. In this context, US20230062382A1 discloses a device composed by a tray receptacle and multiple culture vessels, each capable of supporting the culture of organoids. More particularly, each culture chamber can host a singular cell culture that can be in the form of organoid culture. The multiple culture vessels can be assembled longitudinally and are provided with culture chamber that is in fluidic connection (achieved via small openings) with reservoirs and an additional space called “slot”. Despite fluidic connection may take place between the reservoir and the culture chamber, this connection concerns each singular culture chamber. Therefore, no fluidic connection P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH is established between different culture vessels, meaning that no fluidic connection may take place between different cell cultures (e.g., between adjacent culture chambers). Therefore remains unsatisfied the need of a culture module having a variety of wells, each of these capable of supporting an individual cell culture, wherein a fluidic connection (passive or pump-mediated) can take place between different wells, therefore between different culture chambers. The possibility to have a fluidic connection among different wells belonging to different culture modules is important to allow the flow of cytokines and cells (e.g. immune cells) between the different cell cultures. US2018216057A1 describes a device able to support the culture of tissue engineered constructs, namely on the remit of cardiac tissue engineering. More particularly, the disclosed device involves a tray receptable comprising a substrate with a top surface having multiple wells, where each well can host a single frame. Each single frame is provided with two small housing spaces where clips are placed respectively. Clips are then used in order to grasp tissue samples (e.g., in the form of laser-cut decellularized tissue, hydrogels, etc.). Overall, the housing spaces present in the frame were designed to host the clips, rather than being spaces to develop cell cultures. Each frame can host a single tissue sample (held between the two clips) and no fluidic connection has been described to coculture multiple tissue samples together. No crosstalk between different wells is possible within each frame (or module) as the latter supports the culture of one and single scaffold. In addition, no possible fluidic connection – and therefore biological co-culture among multiple frames – has been described. EP3115449A1 discloses a device suitable for supporting dynamic culture of 2D and 3D biological models. The described culture modules containing multiple culture wells disposed in a row. Each culture has its own lid – this was done to safely handle each well independently, while keeping the other (that still have their own personal lids) safe from any source of contamination. However, fluidic connection within wells leading to the same culture module is not supported. In fact, fluidic connection is allowed only along a first of the two axes of the culture module, while this is not allowed along the other of the two axes of the culture module. Hence, in the device of EP3115449A1, is not possible to fluidically connect each-and-every well P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH present in the plate, but only wells connected along the first of the two cited axes. A full fluidic connection and dynamic culture across different wells of different modules is, thus, not guaranteed. KR20230007120A relates to a modular multiwell plate device capable of hosting different culture modules. This was designed in order to handle multiple samples in a smarter and more efficient way compared to what is currently done with standard (fixed-geometry) multiwell plates. The described device includes a receptacle that is a that is provided with internal partition so that the overall space is divided into regions delimited by planes parallel to the horizontal or vertical edge of the plate itself. Each reference space can host several culture modules or inserts (comprising one or two wells). The culture modules reported in KR20230007120A do not allow any fluidic communications between the wells present in each module. OBJECTS AND SUMMARY OF THE INVENTION A first object of the present invention is, therefore, to provide a bioreactor that is customizable to the different experiments. To this aim, the bioreactor device according to the present invention comprises: - a plurality of modular elements, each of said modular elements comprising at least one well configured to house a biological culture; and - a receptacle configured to house the plurality of modular elements, the receptacle comprising: ^ a tray; and ^ a lid configured for closing the tray. The modular elements are configured to be reversibly placed inside the receptacle in such a way that the at least one well of one modular element is insulated from the at least one well of another modular element. More particularly, the receptacle includes housing spaces that are designed complementarily to the shape of culture modules and the sidewall of the receptacle is provided with a plurality of slots for the insertion of the modular elements. This feature enables users to remove and / or add culture modules in the bioreactor system at any time. Moreover, each module can be designed with wells and / or P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH housing spaces with different geometry and dimensions. As non-limiting examples, the wells can have a rectangle or square, or rhombus and or triangle shape profile and can have a bottom shaped as a flat surface or as a convex multifaceted surface. This could be translated in a “Build your own plate” approach. In this way, it is possible to assemble the bioreactor with as many modules (thus, several wells) as needed during culture experiments reducing the number of unused wells. The same object is also reached by a kit according to the present invention for use in a bioreactor comprising: - at least one modular element, each of said modular elements comprising at least one well configured to house a biological culture; and - a receptacle configured to house the plurality of modular elements and comprising: ^ a tray; and ^ a lid configured for closing the tray. As previously mentioned about the bioreactor, the modular elements are configured to be reversibly placed inside the tray in such a way that the at least one well of one modular element is insulated from the at least one well of another modular element. A second object of the present invention is, then, to provide a bioreactor that allows users to modify wells’ interconnections, by properly changing the way in which wells are hydraulically connected. To this aim the modular elements of the bioreactor or the kit of the present invention can comprise wells interconnected by a septum and the bioreactor can further comprise a clip insert for each couple of interconnected wells. The clip inserts are configured to be reversibly placed into the septum in such a way to prevent the communication between the wells of the couple. A further object of the present invention is to provide a bioreactor that, in addition to be modular, allows for dynamic culture. To this aim, each culture module of the bioreactor or of the kit according to the present invention, can be hydraulically connected to a means of actuation to support both continuous and semicontinuous recirculation of culture medium and, thus, of cells. The connection is possible because P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH each of the plurality of modular elements is provided with holes to host hydraulic adapters and elastomeric O-rings (or any other kind of suitable retention gasket) to secure hydraulic coupling between the modular element and the adapters. These and further features of the present invention will be made clearer by reading the following detailed description, relating to some preferred embodiments of the present invention, to be considered by way of a non-limiting example of the more general concepts claimed. BRIEF DESCRIPTION OF THE DRAWINGS The following description refers to the accompanying drawings, in which: - Figure 1 is an exploded view of a first embodiment of the device of the present invention; - Figure 2 is a perspective view of a detail of the device of the present invention, said detail being relative to the base element of the device; - Figure 3 is a perspective view of a detail of the first embodiment of the device of the present invention, said detail being relative to the lid of said first embodiment; - Figure 4 is a perspective view of a detail of the first embodiment of the device of the present invention, said detail being relative to the receptacle of said first embodiment; - Figure 5 is an exploded view of a second embodiment of the device of the present invention; - Figure 6 is a perspective view of a detail of the second embodiment of the device of the present invention, said detail being relative to the lid of said second embodiment; - Figure 7 is an exploded view of a detail of the second embodiment of the device of the present invention, said detail being relative to the lid of said second embodiment; - Figure 8 is a perspective view of a detail of the second embodiment of the device of the present invention, said detail being relative to the receptacle of said P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH second embodiment; - Figure 9a is an exploded view of a modular element of a first and of the second embodiment of the device of the present invention; - Figure 9b is a schematic representation of the mechanism of recirculation of the culture medium in a longitudinal section of a modular element of the device of the present invention; - Figure 10 is a schematic representation of the mechanism of recirculation of the culture medium in the device of the present invention when hydraulically connected to actuators; - Figure 11a is a top view of a modular element of a third embodiment of the device of the present invention; - Figure 11b is a top view of a modular element of a fourth embodiment of the device of the present invention; - Figure 11c is a top view of a modular element of a first embodiment of the device of the present invention; - Figure 12a is a top view of a modular element of a fifth embodiment of the device of the present invention; - Figure 12b is a top view of a modular element of a sixth embodiment of the device of the present invention; - Figure 12c is a top view of a modular element of a seventh embodiment of the device of the present invention; - Figure 13a is a top view of a modular element of an eighth embodiment of the device of the present invention; - Figure 13b is a top view of a modular element of a ninth embodiment of the device of the present invention; - Figure 13c is a top view of a modular element of a tenth embodiment of the device of the present invention; - Figure 14a is a perspective view of a modular element of an eleventh embodiment of the device of the present invention; - Figure 14b is a top view of a modular element of an eleventh embodiment of the P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH device of the present invention; - Figure 14c is a longitudinal section of a modular element of an eleventh embodiment of the device of the present invention; - Figure 15a is a perspective view of a modular element of a twelfth embodiment of the device of the present invention; - Figure 15b is a top view of a modular element of a twelfth embodiment of the device of the present invention; - Figure 15c is a longitudinal section of a modular element of a twelfth embodiment of the device of the present invention; - Figure 16a is a perspective view of an assembly comprising the modular element of the third embodiment of the device of the present invention and a clip insert; - Figure 16b is an exploded view of an assembly comprising the modular element of the third embodiment of the device of the present invention and a clip insert; - Figure 17a is a perspective view of a detail of the device of the present invention relating to the clip insert; - Figure 17b is a side view of a detail of the device of the present invention relating to the clip insert; - Figure 17c is a bottom view of a detail of the device of the present invention relating to the clip insert; - Figure 17d is a front view of a detail of the device of the present invention relating to the clip insert; - Figure 18a is a perspective view of an assembly comprising the modular element of the sixth embodiment of the device of the present invention and a clip insert; - Figure 18b is an exploded view of an assembly comprising the modular element of the sixth embodiment of the device of the present invention and a clip insert; - Figure 19a is a perspective view of an assembly comprising the modular element of the fifth embodiment of the device of the present invention and two clip inserts; P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH - Figure 19b is a perspective view of an assembly comprising the modular element of the fifth embodiment of the device of the present invention and a clip insert in a first position; - Figure 19c is a perspective view of an assembly comprising the modular element of the fifth embodiment of the device of the present invention and a clip insert in a second position; - Figure 19d is an exploded view of an assembly comprising the modular element of the eighth embodiment of the device of the present invention and two clip inserts; - Figure 20 is a perspective view of a modular element of a thirteenth embodiment of the device of the present invention; - Figure 21a is a schematic representation of a first mechanism of cross-talk recirculation of the culture medium in the device of the present invention ; and - Figure 21b is a schematic representation of a second mechanism of cross-talk recirculation of the culture medium in the device of the present invention. DETAILED DESCRIPTION OF THE INVENTION Referring to Figures 1, 2, 3, 4, 9a, 9b, 10, 11c, 21a, 21b a first embodiment of the bioreactor device of the present invention comprises: - a plurality of modular elements (1, 1’, 1’’), each of said modular elements (1, 1’, 1’’) comprising four wells (14’’) configured to house a biological culture; and - a receptacle (100) configured to house the plurality of modular elements (1, 1’, 1’’), the receptacle (100) comprising: ^ a tray (101); and ^ a lid (102) configured for closing the tray (101); The modular elements (1, 1’, 1’’) are configured to be reversibly placed inside the receptacle (100) in such a way that one well (14’’) of one modular element (1, 1’, 1’’) is insulated from one well of another modular element (1, 1’, 1’’). The tray (101) P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH comprises: - a base surface (50); and - a sidewall (60) extending from the periphery of the base surface (50), said sidewall (60) being provided with a plurality of slots (21, 21’, 21’’) for the insertion of the modular elements (1, 1’, 1’’). The four wells (14’’) have a square profile and a flat bottom. The receptacle (100) and / or the modular elements (1, 1’, 1’’) are made of a 3D printing resin or of a thermoplastic polymer, said thermoplastic polymer being selected among the group consisting in thermoplastics for 3D printing and thermoplastics for subtractive manufacturing. Each of the four modular elements (1, 1’, 1’’) is configured to be hydraulically connected to actuators (300) allowing for the recirculation of culture medium. Each of the four modular elements (1, 1’, 1’’) is provided with holes (74) to host hydraulic adapters and elastomeric O-rings (64, 64’) to secure hydraulic coupling between the modular element and the adapters. The dimension of the base element and the lid were defined in order to create narrow interstitial space (e.g., 200 µm for three spatial directions x,y,z) according to Pasteur’s tortuous path theory. In this way, and thanks to the presence of the pillars (55, 55’, 65, 65’) at each corner of the tray (101), it is possible to allow oxygen supply and prevention from culture contamination at the same time. This kind of coupling allows the realization of 3D clearance between the base element and the lid, thus achieving the aforementioned aims. Referring to Figures 2, 5, 6, 7, 8, 9a, 9b, 10, 11c, 21a, 21b a second embodiment of the bioreactor device of the present invention comprises: - a plurality of modular elements (1, 1’, 1’’), each of said modular elements (1, 1’, 1’’) comprising four wells (14’’) configured to house a biological culture; and - a receptacle (200) configured to house the plurality of modular elements (1, 1’, 1’’), the receptacle (200) comprising: ^ a tray (101); and ^ a lid (202) configured for closing the tray (101); The modular elements (1, 1’, 1’’) are configured to be reversibly placed inside the P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH receptacle (200) in such a way that the one well (14’’) of one modular element (1, 1’, 1’’) is insulated from the at least one well of another modular element (1, 1’, 1’’). The tray (101) comprises: - a base surface (50); and - a sidewall (60) extending from the periphery of the base surface (50), said sidewall (60) being provided with a plurality of slots (21, 21’, 21’’) for the insertion of the modular elements (1, 1’, 1’’). The four wells (14’’) have a square profile and a flat bottom. The tray (101) and / or the modular elements (1, 1’, 1’’) are made of a 3D printing resin or of a thermoplastic polymer, said thermoplastic polymer being selected among the group consisting in thermoplastics for 3D printing and thermoplastics for subtractive manufacturing. The lid (202), instead, comprises a first transparent layer of polycarbonate (203), a second gasket layer (203’) of silicone and a third frame layer (203’’) of polycarbonate. Each of the four modular elements (1, 1’, 1’’) is configured to be hydraulically connected to actuators (300) allowing for the recirculation of culture medium. Each of the four modular elements (1, 1’, 1’’) is provided with holes (74) to host hydraulic adapters and elastomeric O-rings (64, 64’) to secure hydraulic coupling between the modular element and the adapters. The dimension of the base element and the lid were defined in order to create narrow interstitial space (e.g., 200 µm for three spatial directions x,y,z) according to Pasteur’s tortuous path theory. In this way, and thanks to the presence of the pillars (55, 55’, 65, 65’) at each corner of the tray (101), it is possible to allow oxygen supply and prevention from culture contamination at the same time. Referring to Figures 2, 9b, 11a, 16a, 16b, 17a, 17b, 17c and 17d, a third embodiment of the bioreactor device of the present invention is identical to the first or second embodiment of the present invention except that the modular elements (2) comprise only two wells (14, 14’) configured to house a biological culture. The two wells (14, 14’) have a square profile and have a flat bottom. The two wells (14, 14’), are interconnected by a septum (54) and the bioreactor device comprises also a clip insert (41) for the couple of the interconnected wells (14, 14’), said clip insert (41) being configured to be reversibly placed into the septum (54) in such a way to prevent the communication P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH between the wells (14, 14’) of the couple. The clip insert (41) has two longitudinal fins that allow it to remain in position inside the modular element (2). Referring to Figures 2, 9b and 11b, a fourth embodiment of the bioreactor device of the present invention is identical to the first or second embodiment of the present invention except that the modular element (3) comprises three wells (14’’’) configured to house a biological culture. The three wells (14’’’) have a square profile and have a flat bottom. Referring to Figures 2, 9b, 12a, 17a, 17b, 17c, 17d, 19a, 19b, 19c and 19d, a fifth embodiment of the bioreactor device of the present invention is identical to the first or second embodiment of the present invention except that the modular element (5) comprises three wells (16, 16’, 16’’) configured to house a biological culture. The three wells (16, 16’, 16’’) have a rectangular profile and a flat bottom. Two (16, 16’’) of the three wells (16, 16’, 16’’) have the same dimensions, whereas the third well (16’) is larger than the first two (16, 16’’) and is placed in the middle of the first two (16, 16’’). Both the couples formed by the first (16) and the third (16’) well and the third (16’) and the second (16’’) well are interconnected by a septum (56, 56’). The bioreactor device comprises two clip inserts (41, 41’), one for each couple of interconnected wells (16, 16’), (16’,16’’), said clip inserts (41, 41’) being configured to be reversibly placed into the septa (56, 56’) in such a way to prevent the communication between the wells (16, 16’,16’’) of the couple. The clip inserts (41, 41’) have two longitudinal fins that allow it to remain in position inside the modular element (5). The modular element (5) of the fifth embodiment of the present invention represents a potential solution for use where there is a need for a larger well (e.g., to accommodate a construct or tissue section) and two smaller 'reservoirs'. Referring to Figures 2, 9b, 12b, 17a, 17b, 17c, 17d, 18a and 18b, a sixth embodiment of the bioreactor device of the present invention is identical to the first or second embodiment of the present invention except that the modular elements (6) comprise only two wells (17, 17’) configured to house a biological culture. The two wells (17, 17’) have a rectangular profile, different dimensions and a flat bottom. The two wells (17, 17’), are interconnected by a septum (57) and the bioreactor device comprises also a P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH clip insert (41) for the couple of the interconnected wells (17, 17’), said clip insert (41) being configured to be reversibly placed into the septum (57) in such a way to prevent the communication between the wells (17, 17’) of the couple. The clip insert (41) has two longitudinal fins that allow it to remain in position inside the modular element (6). Also, the modular element (6) of the sixth embodiment of the present invention represents a potential solution for use where there is a need for a larger well (e.g. to accommodate a construct or tissue section) and a smaller 'reservoir'. Referring to Figures 2, 9b and 12c, a seventh embodiment of the bioreactor device of the present invention is identical to the first or second embodiment of the present invention except that the modular element (7) comprises two wells (18, 18’) having a triangular profile and a third well having a square profile in the middle of the two triangular wells (18, 18’). All the wells (18, 18’) have a flat bottom. The alternation of converging and diverging sections implies an improvement from a fluid-dynamic point of view during dynamic culture, because it has the benefit of better guiding the culture medium from the inlet to the outlet of the module. This promotes an increase in mass transport and a reduction in stagnation zones (with possible deposits of biological material). In the seventh embodiment of the bioreactor device, the central rectangular well has an area greater than the triangular wells and, therefore, also the modular element (7) of the seventh embodiment of the present invention represents a potential solution for use where there is a need for a larger well and two smaller 'reservoirs'. Referring to Figures 2, 9b and 13a, an eighth embodiment of the bioreactor device of the present invention is identical to the first or second embodiment of the present invention except that the modular element (8) comprises two wells (15, 15’) having a rhomboidal profile. All the wells (15, 15’) have a flat bottom. Referring to Figures 2, 9b and 13b, a ninth embodiment of the bioreactor device of the present invention is identical to the first or second embodiment of the present invention except that the modular element (9) comprises three wells (15’’) having a rhomboidal profile. All the wells (15’’) have a flat bottom. Referring to Figures 2, 9b and 13c, a tenth embodiment of the bioreactor device of the P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH present invention is identical to the first or second embodiment of the present invention except that the modular element (10) comprises five wells (15’’’) having a rhomboidal profile. All the wells (15’’’) have a flat bottom. In all the embodiments from eighth to tenth, the rhomboid wells show an improvement from a fluid-dynamic point of view during dynamic culture because of the alternation of converging and diverging sections has the benefit of better guiding the culture medium fluid from the inlet to the outlet of the module. Referring to Figure 14a, 14b and 14c, an eleventh embodiment of the bioreactor device of the present invention is identical to the third embodiment of the present invention except that the two wells of the modular element (2’) have a bottom shaped as a convex multifaceted surface (19) with a hexagonal geometry. Referring to Figure 15a, 15b and 15c, a twelfth embodiment of the bioreactor device of the present invention is identical to the third embodiment of the present invention except that the two wells of the modular element (2’’) have a bottom shaped as a convex multifaceted surface (19’) with an octagonal geometry. In both the eleventh and twelfth embodiments, the presence of depressions instead of a completely flat bottom surface allows to prevent the cells from adhesion to the bottom of the wells. In this way, the potential construct, not shown in the figure, is conditioned by the culture medium on both the inferior and superior portions with respect to a plane parallel to the supporting surface of the device (i.e. the plane indicated with the letter (x) in Figures 14c and 15c). Referring to Figure 3, 6, 20, a thirteenth embodiment of the present invention comprises a tray (101) and a lid (102, 202) according the first or the second embodiment of the bioreactor described above and four modular elements (4, 8, 2’, 1). The first modular element (4) comprises only one well with a rectangular profile and a flat bottom, the second modular element (8) comprises two wells with a rhomboidal profile, the same dimensions and a flat bottom, the third modular element (2’) comprises two wells with a square profile, the same dimensions and a bottom shaped as a convex multifaceted surface and, finally, the fourth modular element (1) comprises four wells with a square profile, the same dimensions and a flat bottom. By providing P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH modular elements with wells having different geometry and dimension, it becomes possible to assemble the bioreactor as needed during experiments and, thus, to reduce the number of unused wells, according the “Build your own plate” approach cited above. EXAMPLES Example 1 A possible application of the bioreactor device of the present invention is the simulation of the invasion of tumour constructs by immune cells in order to study immune cell-mediated tumour progression mechanisms and its possible implications. The experiment can be performed according to the following steps: 1. Initial condition: the clip insert is inserted and fixed within the modular element to prevent the initial fluidic connection between wells (Figure 18a). 2. A construct / biopsy / section of original tumour tissue with its own culture medium is placed in larger well. 3. Immune cells (derived from the same source as the previous construct / biopsy or from alternative sources) resuspended in their own culture medium are placed in the smaller wells. 4. Removal of the insert (Figure 18b). This allows fluidic connection of the two wells. We might expect immune cells in the smaller wells to migrate to the larger well to invade the tumour mass. Example 2 A second possible application of the bioreactor device of the present invention is the study of the agonistic or antagonistic effect on the invasion of the tumour model. The experiment can be performed according to the following steps: 1. Initial condition: the two inserts are inserted and fixed within the modular element to prevent the initial fluidic connection between wells (Figure 19a). P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH 2. A construct / biopsy / section of original tumour tissue with its own culture medium is placed in larger central well. The tumour model is treated with molecules that can affect immune cell activity and migration. 3. Immune cells (derived from the same source as the previous construct / biopsy or from alternative sources) resuspended in their own culture medium are placed in the smaller wells / reservoirs. 4. Removal of the insert (Figure 19d). This allows fluidic connection of the two wells. We might expect changes in the invasion of immune cells into the tumour model depending on the type of molecules added to the culture. Example 3 A possible application of the present bioreactor device could concern the culture of multiple biological replicates in the same culture conditions (Figure 10, 21a, 21b). This would be achieved by hydraulically connecting multiple culture modules, so to allow the establishment of a culture setting involving a higher number of replicates. This setting would allow the study of the cross-talk among different constructs / biopsies / tissue samples isolated from the same tissue source or from multiple sources (Figure 21a, 21b).

Claims

P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH CLAIMS 1. Bioreactor device comprising: - a plurality of modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10), each of said modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) comprising at least two wells (14, 14’, 14’’, 14’’’, 15, 15’, 15’’, 15’’’, 16, 16’, 16’’, 17, 17’, 18, 18’) configured to house a biological culture; and - a receptacle (100, 200) configured to house the plurality of modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10), the receptacle (100, 200) comprising: ^ a tray (101); and ^ a lid (102, 202) configured for closing the tray (101); said modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) being configured to be reversibly placed inside the receptacle (100, 200) in such a way that the at least one of the two wells (14, 14’, 14’’, 14’’’, 15, 15’, 15’’, 15’’’, 16, 16’, 16’’, 17, 17’, 18, 18’) of one modular element (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) is insulated from at least one of the two wells (14, 14’, 14’’, 14’’’, 15, 15’, 15’’, 15’’’, 16, 16’, 16’’, 17, 17’, 18, 18’) of another modular element (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10); characterized in that the at least two wells (14, 17, 16, 16’, 14’, 17’,16’’) of one modular element (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) are interconnected by a septum (54, 56, 56’, 57); and in that the bioreactor device comprises a clip insert (41, 41’) for each couple of interconnected wells (14, 17, 16, 16’, 14’, 17’, 16’’), said clip insert (41, 41’) being configured to be reversibly placed into the septum (54, 56, 56’, 57) in such a way to prevent the communication between the wells (14, 17, 16, 16’, 14’, 17’,16’’) of the couple. -1-P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH 2. Bioreactor device according to claim 1, wherein said tray (101) comprises: - a base surface (50); and - a sidewall (60) extending from the periphery of the base surface (50), said sidewall (60) being provided with a plurality of slots (21, 21’, 21’’) for the insertion of the modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10).

3. Bioreactor device according to any of the preceding claims, wherein the at least a well (14, 14’, 14’’, 14’’’, 15, 15’, 15’’, 15’’’, 16, 16’, 16’’, 17, 17’, 18, 18’) has a shape profile selected from the group consisting in: a rectangle, a square, a rhombus and a triangle.

4. Bioreactor device according to any of the preceding claims, wherein the at least a well (14, 14’, 14’’, 14’’’, 15, 15’, 15’’, 15’’’, 16, 16’, 16’’, 17, 17’, 18, 18’) has a flat bottom.

5. Bioreactor device according to any of the claims from 1 to 3, wherein, the at least a well (14, 14’, 14’’, 14’’’, 15, 15’, 15’’, 15’’’, 16, 16’, 16’’, 17, 17’, 18, 18’) has a bottom shaped as a convex multifaceted surface (19, 19’).

6. Bioreactor device according to any of the preceding claims wherein the tray (101) is provided with a pillar (55, 55’, 65, 65’) for each of its corners.

7. Bioreactor device according to any of the preceding claims wherein the receptacle (100, 200) and / or the modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) are made of a 3D printing resin.

8. Bioreactor device according to any of the claims from 1 to 6 wherein the receptacle (100, 200) and / or the modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) are made of a thermoplastic polymer, said thermoplastic polymer being selected among the group consisting in thermoplastics for 3D printing and thermoplastics for subtractive manufacturing.

9. Bioreactor device according to any of the preceding claims wherein each of the -2-P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH plurality of modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) is configured to be hydraulically connected to actuators (300) allowing for the recirculation of culture medium.

10. Bioreactor device according to claim 9 wherein each of the plurality of modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) is provided with holes (74) to host hydraulic adapters and elastomeric O-rings (64, 64’) to secure hydraulic coupling between the modular element (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) and the adapters.

11. Kit for use in a bioreactor comprising: - at least one modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10), each of said modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) comprising at least one well (14, 14’, 14’’, 14’’’, 15, 15’, 15’’, 15’’’, 16, 16’, 16’’, 17, 17’, 18, 18’) configured to house a biological culture; and - a receptacle (100, 200) configured to house the plurality of modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) and comprising: ^ a tray (101); and ^ a lid (102, 202) configured for closing the tray (101); characterized in that said modular elements (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) are configured to be reversibly placed inside the tray (101) in such a way that the at least one well (14, 14’, 14’’, 14’’’, 15, 15’, 15’’, 15’’’, 16, 16’, 16’’, 17, 17’, 18, 18’) of one modular element (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) is insulated from the at least one well of another modular element (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10).

12. Kit according to the claim 11 wherein at least one of the plurality of the modular element (1, 1’, 1’’, 2, 2’, 2’’, 3, 4, 5, 6, 7, 8, 9, 10) comprises at least two wells (14, 15, 16, 16’, 14’, 17’, 16’’), said two wells (14, 15, 16, 16’, 14’, 17’, 16’’) being interconnected by a septum (54, 56, 56’, 57). -3-P6452PC00 of:POLITECNICO DI MILANO FOUNDATION FOR LIVER RESEARCH 13. Kit according to the claim 12 comprising a clip insert (41, 41’) for each couple of interconnected wells (14, 15, 16, 16’, 14’, 17’,16’’), said clip insert (41, 41’) being configured to be placed into the septum (54, 56, 56’, 57) in such a way to prevent the communication between the wells (14, 15, 16, 16’, 14’, 17’,16’’) of the couple. -4-

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