Multilayer microfluidic device for cell culture

Abstract

A device for culturing biological cell structures, comprising: a first level comprising a plurality of microfluidic channels comprising a pair of microfluidic channels; a second level, different from the first level, comprising one or more hydrogel compartments for hosting a hydrogel, wherein each hydrogel compartment is in fluid communication with said pair of microfluidic channels; and a substrate forming a wall of a cell culture compartment for hosting one or more biological cell structures, said culture compartment comprising one or more biological cell structure docking sites, each docking site comprising a through-hole having a top surface, said through-hole ensuring fluid communication between the cell culture compartment and the hydrogel compartment, said through-hole being situated laterally between the pair of microfluidic channels; and a docking structure for docking a three-dimensional biological cell structure so that it is in fluidic communication with the through-hole.

Description

[0001] Multilayer microfluidic device for cell culture

[0002] Field of the Invention

[0003] The present invention relates to the field of microfluidic devices for biological cell culture, and more specifically to devices for culturing three-dimensional biological cell structure.

[0004] Background of the Invention

[0005] In recent years, the field of regenerative medicine and biomedical research has witnessed significant advancements with the development of human three-dimensional biological cell structure technology such as organoids. Organoids are three-dimensional biological cell structures grown from stem cells that mimic the architecture and functionality of real organs. They have emerged as valuable tools for disease modeling, drug discovery, and understanding complex biological processes in a controlled laboratory setting. By replicating human physiology more accurately than traditional two-dimensional biological cell cultures or animal models, organoids offer the potential to enhance preclinical studies and reduce the reliance on animal testing.

[0006] Despite the promise of three-dimensional biological cell structure technology, several challenges persist in limiting its full potential. One of the primary obstacles is achieving effective vascularization within these structures. Vascularization supplies nutrients and oxygen and removes metabolic waste, which supports the growth, maturation, and sustained viability of three-dimensional biological cell structures in vitro. Without adequate vascular networks, such structures may suffer from issues related to nutrient diffusion limitations, leading to cell death or incomplete development, particularly in the core regions of larger three-dimensional biological cell structures.

[0007] Another challenge lies in the spatial distribution and organization of three-dimensional biological cell structures during cultivation. Random organization can result in variability between three-dimensional biological cell structures, affecting the consistency and reproducibility of experimental results.

[0008] Control over the microenvironment, including fluid dynamics and shear stress, is also an area of concern. Inadequate regulation of flow directionality and fluid dynamics may lead to instability in three-dimensional biological cell structure cultures, with a tendency for regression or loss of functionality over time. Furthermore, replicating the hierarchicalstructure of vascular networks found in vivo remains a complex task. Without this hierarchy, it is challenging to emulate the microvascular circulation of organs and their response to stimuli.

[0009] These challenges highlight the limitations of current methodologies in three-dimensional biological cell structure culture and vascularization. While strides have been made, there is still a considerable gap in replicating the intricate vascular systems and controlled environments necessary for three-dimensional biological cell structures to fully model human physiological and pathological processes. Consequently, there is a need for further advancements in this field to address at least some of these challenges, enhancing the utility and applicability of three-dimensional biological cell structure technology in research and clinical contexts.

[0010] Summary of the Invention

[0011] It is an object of embodiments of the present invention to provide a good microfluidic device and method for culturing vascularized three-dimensional biological cell structures. This objective is accomplished by the aspects of the present invention.

[0012] In a first aspect, the present invention relates to a microfluidic device for culturing three-dimensional biological cell structures, comprising:

[0013] - a first level comprising a plurality of microfluidic channels comprising a pair of microfluidic channels,

[0014] - a second level, different from the first level, comprising one or more hydrogel compartments for hosting a hydrogel, wherein each hydrogel compartment is in fluid communication with said pair of microfluidic channels, and

[0015] - a substrate having a top surface, said substrate forming a wall of a cell culture compartment for hosting one or more three-dimensional biological cell structures, said cell culture compartment comprising one or more three-dimensional biological cell structure docking sites, each docking site comprising:

[0016] - a through-hole having a top surface having an area, said through-hole ensuring fluid communication between the cell culture compartment and a hydrogel compartment, said through-hole being situated laterally between the pair of microfluidic channels, and

[0017] - a docking structure for docking a three-dimensional biological cell structure so that the three-dimensional biological cell structure is in fluidic communication with the through-hole.In embodiments, the second level may be disposed above the first level. This configuration allows for efficient stacking of the microfluidic channels and hydrogel compartment(s).

[0018] In embodiments, the substrate may be comprised in a third level, disposed above the second level, itself above the first level, wherein the substrate forms the lowest part of the third level and a bottom wall of the cell culture compartment. This three-level architecture enables optimal spatial arrangement of the components.

[0019] In embodiments, the docking structure may be for docking the three-dimensional biological cell structure so that the three-dimensional biological cell structure covers at least partly (e.g., completely) the through-hole. This ensures proper fluidic communication between the three-dimensional biological cell structure and a hydrogel compartment.

[0020] In embodiments, the three-dimensional biological cell structure may be a type of three-dimensional biological cell structure that requires vascularization to survive. The device is particularly suited for culturing cell structures with high vascularization needs.

[0021] In embodiments, the three-dimensional biological cell structure may be selected from organoids, spheroids, and three-dimensional tissue engineering constructs. These are non-limiting examples of relevant biological structures that can be cultured in the device.

[0022] In embodiments, the docking structure may be configured such that, if one closes its uppermost boundary by a horizontal plane, one or more openings are comprised in its outermost lateral boundary. This configuration facilitates lateral access to the docked three-dimensional biological cell structure.

[0023] In embodiments, the innermost lateral boundary of the docking structure may be at all points laterally separated from the top surface of the through-hole by a distance of at most 10%, preferably at most 5%, yet more preferably at most 2% of the largest lateral dimension of the top surface of the through-hole. Limiting this separation distance ensures compact docking.

[0024] In embodiments, the docking structure may comprise one or more protrusions projecting from the top surface of the substrate. The protrusions aid in securely docking the three-dimensional biological cell structure.

[0025] In embodiments, the one or more protrusions may be projecting vertically from the top surface of the substrate. Vertical protrusions provide optimal structural support.

[0026] In embodiments, the one or more protrusions may be a plurality of spaced-apart protrusions. Spaced protrusions allow for fluid access between them.In embodiments, the one or more protrusions may be a plurality of protrusions in a number of at least 3, preferably from 3 to 10, more preferably from 4 to 8, yet more preferably from 5 to 7, such as 6. This range of protrusion numbers has been found to provide good docking performance.

[0027] In embodiments, the one or more protrusions may be regularly spaced. Regular spacing provides a uniform docking interface.

[0028] In embodiments, the plurality of spaced protrusions may be surrounding the through-hole. Surrounding the through-hole with protrusions facilitates centering the three-dimensional biological cell structure over it.

[0029] In embodiments, all protrusions may be at a same distance from the through-hole. Equidistant protrusions provide balanced structural support.

[0030] In embodiments, the protrusions may be pillars. Pillars are simple yet effective protrusion structures.

[0031] In embodiments, the largest distance between any two neighboring protrusions may be from 100 to 200 pm. This distance range allows adequate three-dimensional biological cell structure support while permitting fluid access.

[0032] In embodiments, the pair of microfluidic channels may comprise an inlet channel for introducing matter into the one or more hydrogel compartments and an outlet channel for removing matter from the one or more hydrogel compartments. Dedicated inlet and outlet channels enable efficient fluid exchange.

[0033] In embodiments, the pair of microfluidic channels may be arranged in parallel. A parallel arrangement provides a streamlined fluid circuit.

[0034] In embodiments, the device may comprise at least two docking sites, preferably from 10 to 20 docking sites, situated between said pair of microfluidic channels. Multiple docking sites allow simultaneous culturing of several three-dimensional biological cell structures. In embodiments, the at least two docking sites may form an array of docking sites aligned in two dimensions. In embodiments, these at least two docking sites may be distributed among a plurality of hydrogel compartments. For instance, the number of docking sites situated between said pair may be equal to the number of hydrogel compartments. This corresponds to a situation where each hydrogel compartment portion present between the pair of microfluidic channels has exactly one docking site with its associated through-hole. As another example, the number of docking sites situated between said pair may be equal to an integer multiple of the number of hydrogel compartments. This corresponds to a situation where each hydrogel compartment portion present between the pair of microfluidicchannels has multiple docking sites, for example two, three, or four docking sites, each with its associated through-hole. In such a situation, biological three-dimensional biological cell structure of a same type may occupy each of the multiple docking sites or different three-dimensional biological cell structure may be present at the multiple docking sites.

[0035] In embodiments, the microfluidic channels of the pair may be parallel and said at least two docking sites may have their through-holes aligned in a first direction parallel to the direction of the microfluidic channels of the pair. Aligned through-holes facilitate uniform fluid delivery to the three-dimensional biological cell structures.

[0036] In embodiments, the plurality of microfluidic channels may comprise at least three microfluidic channels forming a first and a second pair of microfluidic channels sharing a central microfluidic channel, a first and a second series of docking sites, wherein said first series of docking sites is laterally between the microfluidic channels of the first pair, and wherein said second series of docking sites is laterally between the microfluidic channels of the second pair. This configuration enables high-density docking site packing.

[0037] In embodiments, the first and second series of docking sites may have their through-holes aligned in the first direction. Alignment in the flow direction promotes uniform fluid access.

[0038] In embodiments, each hydrogel compartment may be oblong and have its longitudinal direction forming an angle, preferably from 80 to 100°, more preferably a 90° angle, with the longitudinal direction of each microfluidic channel of the pair. This allows each hydrogel compartment to intersect with the pair of microfluidic channels, thereby enabling fluidic communication between each hydrogel compartment and each microfluidic channel of the pair.

[0039] In embodiments, each hydrogel compartment is oblong and intersects with each of the microfluidic channels.

[0040] In embodiments, each hydrogel compartment may be in fluid communication with said pair of microfluidic channels via openings having a hydraulic diameter of from 5 to 200 micrometers, preferably 20 to 150 micrometers, more preferably 60 to 130 micrometers. These opening size ranges allow proper hydrogel retention while permitting fluid exchange.

[0041] In embodiments, the docking structure may have an outermost boundary having a maximal length measured parallel to the top surface of the substrate of from 50 to 400 micrometers, preferably from 70 to 300 micrometers, more preferably from 80 to 250 pm. These dimensions are well-suited for typical three-dimensional biological cell structure sizes.In embodiments, the microfluidic channels may have a height of from 50 to 300 micrometers. This height range provides a good balance between fluid flow and device compactness.

[0042] In embodiments, the microfluidic channels may have a width of from 50 to 300 micrometers. This width range accommodates typical fluid flow needs.

[0043] In embodiments, the device may be at least partly fabricated from optically transparent materials so that when three-dimensional biological cell structures are present, they can be observed from the top and / or the bottom of the device. Optical transparency enables monitoring of the three-dimensional biological cell structures.

[0044] In embodiments, the device may be at least partially fabricated from materials having a UV light transmission of at least 80% so as to enable UV-initiated crosslinking of the hydrogel when loaded in the hydrogel compartment.

[0045] In embodiments, the device may be at least partially fabricated from materials selected from optically transparent polymers having a thickness and UV transmission properties that ensure UV light attenuation does not exceed 20% during hydrogel crosslinking processes. Examples of such materials include poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), and cyclic olefin copolymer (COC).

[0046] In embodiments, the device may further comprise a biosensor configured for measuring at least one parameter from a three-dimensional biological cell structure when it is docked at a docking site and / or configured for detecting a substance in the cell culture compartment. Integrated biosensors allow real-time monitoring of the biological environment.

[0047] In embodiments, said biosensor may comprise one or more electrodes. Electrodes are versatile sensors for biological systems.

[0048] In embodiments, the device may further comprise the hydrogel and the three-dimensional biological cell structures.

[0049] In embodiments, the three-dimensional biological cell structures may each be of a type that requires vascularization to survive. The device is particularly suited for culturing cell structures with high vascularization needs.

[0050] In embodiments, each of the three-dimensional biological cell structures may be independently selected from organoids, spheroids, and three-dimensional tissue engineering constructs. These are non-limiting examples of relevant biological structures that can be cultured in the device. Preferably, each of the three-dimensional biologic cell structure is an organoid.In embodiments, the three-dimensional biological cell structures may comprise different three-dimensional biological cell structures.

[0051] In embodiments, the three-dimensional biological cell structures may each be a human organoid independently selected from the group consisting of brain organoids, liver organoids, kidney organoids, cardiac organoids, gut organoids, and tumor organoids. These organoid types are of great biomedical interest, but the present invention is applicable to any organoids.

[0052] In embodiments, the three-dimensional biological cell structures docked on docking sites comprising a through-hole ensuring fluid communication between the cell culture compartment and a same hydrogel compartment, comprise different three-dimensional biological structures. In other words, the three-dimensional biological cell structures in fluid communication with the same hydrogel compartment may comprise different three-dimensional biological structures. For instance, the three-dimensional biological cell structures in fluid communication with the same hydrogel compartment may comprise different human organoids, for instance selected from the group consisting of brain organoids, liver organoids, kidney organoids, cardiac organoids and gut organoids. In embodiments, several or all of these different human organoids (brain organoids, liver organoids, kidney organoids, cardiac organoids, and gut organoids) may be present, thereby forming a "human-on-chip".

[0053] In embodiments, the three-dimensional biological cell structures may be vascularized. Vascularization is enabled by the device and supports long-term cell structure viability.

[0054] In embodiments, the device may further comprise a vascular network within the hydrogel, said vascular network being vascularly connected to the three-dimensional biological cell structures. An integrated vascular network that supplies the three-dimensional biological cell structures greatly enhances their growth and survival.

[0055] In embodiments, the vascular network may comprise anastomosed vasculature (e.g., formed by angiogenesis and / or vasculogenesis) from a hydrogel compartment into the three-dimensional biological cell structure. Angiogenesis and vasculogenesis are effective natural mechanisms for generating vascular networks.

[0056] In a second aspect, the present invention relates to a system comprising a device according to any one of the preceding claims, and a flow controller for applying controlled flow of culture medium through a microfluidic channel of the pair. A flow control system allows optimizing the fluid environment for the three-dimensional biological cell structures.Any feature of this aspect may be as correspondingly described in any embodiment of any other aspect of the present invention.

[0057] In a third aspect, the present invention relates to a method of vascularizing three-dimensional biological cell structures, comprising providing a system as described in the second aspect, introducing endothelial cells and pericytes into each hydrogel compartment, providing three-dimensional biological cell structures at the docking sites, and applying controlled flow of culture medium through the pair of microfluidic channels, thereby vascularizing the three-dimensional biological cell structures. In embodiments, other vascular-supporting cells may also be introduced into each hydrogel compartment.

[0058] In embodiments, the method may further comprise introducing endothelial cells and / or pericytes into the microfluidic channels to endothelialize the channels. In embodiments, other vascular-supporting cells may also be introduced into the microfluidic channels. Endothelialized channels provide a more physiological fluid delivery system.

[0059] In embodiments, the three-dimensional biological cell structures may be introduced as pre-formed three-dimensional biological cell structures. Pre-forming the cell structures allows optimizing their initial composition and organization.

[0060] In embodiments, the three-dimensional biological cell structures may be formed in situ by introducing cell suspensions in contact with the docking sites. In situ formation simplifies the cell seeding process.

[0061] In embodiments, the method may further comprise harvesting the vascularized three-dimensional biological cell structures. Harvesting allows detailed analysis and further use of the cell structures.

[0062] In embodiments, different three-dimensional biological cell structures may be cultured simultaneously at different docking sites. Simultaneous culturing of different cell structure types allows studying their interactions. For instance, organoids of different organs stemming from a same individual may be cultured simultaneously at different docking sites fluidically connected to a same hydrogel compartment. This forms a "human-on-chip". In embodiments, when the three-dimensional biological cell structures in fluid communication with the same hydrogel compartment comprise different three-dimensional biological cell structures, each different three-dimensional biological cell structures may be positioned between a different pair of fluidic channels.

[0063] In alternative embodiments, at least two different three-dimensional biological cell structures may be positioned between the same pair of fluidic channels when in fluid communication with the same hydrogel compartment.In embodiments, the endothelial cells and pericytes (and the other vascular-supporting cells when present) may be derived from human cells, e.g., from human primary cells or human induced pluripotent stem cells. Using human cells makes the method more clinically relevant.

[0064] In embodiments, the three-dimensional biological cell structures may comprise human organoids selected from the group consisting of brain organoids, liver organoids, kidney organoids, cardiac organoids, gut organoids, and tumor organoids. These organoid types are of great biomedical interest.

[0065] In embodiments, applying controlled flow may comprise applying a pressure difference between the first and second microfluidic channel of the pair to generate a pressure gradient within the pair. Pressure gradients can be used to mimic in vivo interstitial flow.

[0066] In embodiments, the method may further comprise introducing compounds or pathogens into the culture medium to assess effects on the three-dimensional biological cell structure. The method is well-suited for drug testing and screening. The method is also well-suited for the determination of pathogen biodistribution and infection routes.

[0067] Any feature of this aspect may be as correspondingly described in any embodiment of any other aspect of the present invention.

[0068] In yet a fourth aspect, the present invention relates to a vascularized three-dimensional biological cell structure produced by the above method. The vascularized cell structures have wide applications in research and therapeutics.

[0069] In embodiments, the three-dimensional biological cell structure may comprise a hierarchical vascular network. A hierarchical vascular network improves cell structure perfusion and function.

[0070] Any feature of this aspect may be as correspondingly described in any embodiment of any other aspect of the present invention.

[0071] It is an advantage of embodiments of the present invention that the microfluidic device allows precise control over microphysiological flow parameters, facilitating the formation of stable and perfusable vascular networks within hydrogel compartments, which extend into and vascularize the three-dimensional biological cell structures.

[0072] It is an advantage of embodiments of the present invention that parallelized three-dimensional biological cell structure docking sites enable simultaneous vascularization of multiple three-dimensional biological cell structures, thus supporting high-throughput three-dimensional biological cell structure culture on-chip.It is an advantage of embodiments of the present invention that the unique design of parallel medium flow channels creates physiologically relevant shear stress and controlled flow directionality, inducing vascular cell specification into a hierarchical cytoarchitecture that emulates arterioles, capillaries, and venules, thereby enhancing microvascular circulation within three-dimensional biological cell structures.

[0073] It is an advantage of embodiments of the present invention that the device enables uniform flow conditions for each three-dimensional biological cell structure, effectively mitigating crosstalk and minimizing the risk of contamination.

[0074] It is a further advantage that each three-dimensional biological cell structure can interact with its dedicated portion of a vascularized hydrogel compartment through its associated through-hole, while the uniform flow conditions through the microfluidic channels ensure consistent nutrient and oxygen delivery to each structure.

[0075] It is an advantage of embodiments of the present invention that the controlled perfusion system ensures the correct distribution of resources to cells, making three-dimensional biological cell structure growth more stable and reliable.

[0076] It is an advantage of embodiments of the present invention that the docking structures allow precise alignment and proper vascularization of docked three-dimensional biological cell structures without jeopardizing their maturation due to mismatches in cellular development.

[0077] It is an advantage of embodiments of the present invention that it reduces random cytoarchitecture by promoting favorable flow properties for cell viability and growth.

[0078] It is an advantage of embodiments of the present invention that the device design enables the formation of an in vivo-mimicking vascular hierarchical structure within three-dimensional biological cell structures, leading to more physiologically relevant models for disease modeling, drug discovery, and regenerative medicine.

[0079] It is an advantage of embodiments of the present invention that the reasonably scaled device footprint accommodates a higher number of three-dimensional biological cell structure dockings, enhancing throughput without sacrificing control over individual three-dimensional biological cell structure environments.

[0080] Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims.The above and other characteristics, features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention. This description is given for the sake of example only, without limiting the scope of the invention. The reference figures quoted below refer to the attached drawings.

[0081] Brief description of the drawings

[0082] Here are the brief descriptions for each figure based on the inventor's input:

[0083] Fig. 1 is a detailed cross-sectional view of a docking site of a microfluidic device according to embodiments of the present invention.

[0084] Fig. 2 is a cross-sectional view of a docking site of a microfluidic device with a three-dimensional biological cell structure docked thereto according to embodiments of the present invention.

[0085] Fig. 3 is a perspective view of a microfluidic device with a plurality of three-dimensional biological cell structures docked thereto according to embodiments of the present invention.

[0086] Fig. 4 is a top view of a docking site of a microfluidic device according to embodiments of the present invention.

[0087] Fig. 5 is a top view of a microfluidic device comprising a plurality of docking sites according to embodiments of the present invention.

[0088] In the different figures, the same reference signs refer to the same or analogous elements.

[0089] Detailed description of Illustrative Embodiments

[0090] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and the relative dimensions do not correspond to actual reductions to practice of the invention.

[0091] Furthermore, the terms first, second, third, and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequence, either temporally, spatially, in ranking, or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstancesand that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0092] It is to be noticed that the term "comprising", used in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. The term "comprising" therefore covers the situation where only the stated features are present (and can therefore always be replaced by "consisting of" in order to restrict the scope to said stated features) and the situation where these features and one or more other features are present. The word "comprising" according to the invention therefore also includes as one embodiment that no further components are present. Thus, the scope of the expression "a device comprising means A and B" should not be interpreted as being limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

[0093] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[0094] Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim.

[0095] Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0096] Furthermore, some of the embodiments are described herein as a method or combination of elements of a method that can be implemented by a processor of a computer system or by other means of carrying out the function. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Furthermore, an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention.

[0097] In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail in order not to obscure an understanding of this description.

[0098] The following terms are provided solely to aid in the understanding of the invention. As used herein, and unless otherwise specified, the term " three-dimensional biological cell structures" refers to three-dimensional assemblies of biological cells cultured in vitro, such as but not limited to organoids, spheroids, and three-dimensional tissue engineering constructs. They typically emulate functions or characteristics of natural tissues or organs.

[0099] As used herein, and unless otherwise specified, the term "hydrogel compartment" refers to a designated space within the microfluidic device intended to contain a hydrogel material. The hydrogel material typically serves as a scaffold or medium to support cell growth, vascularization, or other biological processes within the device.

[0100] As used herein, and unless otherwise specified, the phrase "fluid communication" refers to the condition wherein two or more spaces, compartments, or channels within the device are connected such that fluids can flow between them under operational conditions.

[0101] As used herein, and unless otherwise specified, the term "through-hole" refers to an opening that extends entirely through a layer or substrate within the device, providing a passage that enables fluid communication or physical connection between the cell culture compartment and a hydrogel compartment.As used herein, and unless otherwise specified, the term "docking sites" refers to specific locations within the cell culture compartment configured to accommodate and retain three-dimensional biological cell structures, typically incorporating features like docking structures and aligned with through-holes to enable interaction with a hydrogel compartment.

[0102] As used herein, and unless otherwise specified, the term "docking structure" refers to a physical configuration or feature within the cell culture compartment designed to receive and retain a three-dimensional biological cell structure at a specific location, facilitating interaction with the through-hole and other components of the device.

[0103] As used herein, and unless otherwise specified, the phrase "docking structure comprises one or more protrusions" means that the docking structure includes physical elements that extend from its base or surface, such as pillars or posts, which assist in retaining or supporting the three-dimensional biological cell structures over the through-hole.

[0104] As used herein, and unless otherwise specified, the phrase "projecting vertically from the top surface" refers to protrusions or features that extend upward in a direction perpendicular to the plane of the substrate or base surface from which they originate, effectively providing vertical support or interaction points for the three-dimensional biological cell structures.

[0105] As used herein, and unless otherwise specified, the phrase "protrusions are pillars" indicates that the protrusions have a columnar shape, functioning as vertical supports or spacers within the docking structure, and may have uniform or varying cross-sections, including cylindrical, square, or other geometric shapes.

[0106] As used herein, and unless otherwise specified, the term "hydrogel" refers to a water-swollen, cross-linked polymeric network capable of absorbing water while maintaining its three-dimensional integrity. They may serve as a scaffold or medium for cell culture and supporting the growth and organization of cells, including the development of vascular networks.

[0107] As used herein, and unless otherwise specified, the phrase "a pair of microfluidic channels" refers to two microfluidic channels. They are typically arranged in relation to each other within the device so as to facilitate specific fluid flow patterns or gradients. They are typically on opposite sides of one or more through-holes.

[0108] As used herein, and unless otherwise specified, the phrase "situated laterally between the pair of microfluidic channels" means that a specified structure, such as athrough-hole or docking site, is located in the lateral space between the two microfluidic channels when viewed parallel to the plane of the device's substrate.

[0109] As used herein, and unless otherwise specified, the term "cell culture compartment" refers to a defined space within the device designed for culturing three-dimensional biological cell structures, wherein the substrate forms a wall or boundary of this compartment and includes features such as docking sites and through-holes.

[0110] As used herein, and unless otherwise specified, the term "top surface having an area" refers to the uppermost surface of the through-hole, which possesses a defined area and serves as the interface between the cell culture compartment and the through-hole. The three-dimensional biological cell structure can be positioned so as to at least partly cover this top surface.

[0111] As used herein, and unless otherwise specified, the phrase "covers at least partly and preferably completely the through-hole" means that the three-dimensional biological cell structure is positioned over the through-hole in such a way that it overlies at least a portion of the opening, and preferably spans the entire opening, to maximize interaction with a hydrogel compartment.

[0112] As used herein, and unless otherwise specified, the phrase "type that requires vascularization to survive" refers to three-dimensional biological cell structures that, due to their size, complexity, or metabolic demands, necessitate the development of a vascular network to supply nutrients and oxygen and remove waste products in order to maintain viability and physiological function during in vitro culture.

[0113] As used herein, and unless otherwise specified, the phrase "configured such that, if one closes its uppermost boundary by a horizontal plane, one or more openings are comprised in its outermost lateral boundary" refers to the design of the docking structure where, even when the top is hypothetically sealed by a flat plane, there remain openings or gaps in its side boundaries, allowing for lateral fluid flow or cellular interactions.

[0114] As used herein, and unless otherwise specified, the phrase "innermost lateral boundary of the docking structure" refers to the inner side surfaces of the docking structure that are closest to the center or axis of the docking site, typically surrounding the through-hole and defining the immediate area where the three-dimensional biological cell structure is positioned.

[0115] As used herein, and unless otherwise specified, the term "hydraulic diameter" refers to a calculated dimension used to characterize flow in circular or non-circular openings, defined as four times the cross-sectional area divided by the wetted perimeter, therebydescribing fluid flow characteristics in openings such as those between a hydrogel compartment and microfluidic channels.

[0116] As used herein, and unless otherwise specified, the phrase "device is at least partly fabricated from optically transparent materials" means that the microfluidic device includes components made from materials that allow light transmission, such as glass or transparent polymers like polydimethylsiloxane (PDMS), enabling visual observation or imaging of the three-dimensional biological cell structures from the top and / or bottom of the device.

[0117] As used herein, and unless otherwise specified, the term "biosensor" refers to a component or assembly integrated into the microfluidic device that can detect, measure, or monitor biological parameters or analytes related to the three-dimensional biological cell structures or their environment, potentially including electrical, optical, or chemical sensors such as electrodes, fluorescence detectors, or enzyme-linked sensors.

[0118] As used herein, and unless otherwise specified, the term "vascular network" refers to a network of interconnected vessels formed by endothelial cells and supporting cells like pericytes. It is capable of facilitating the flow of fluids, nutrients, and signaling molecules, and potentially mimicking the hierarchical organization of in vivo blood vessels within the hydrogel and three-dimensional biological cell structures.

[0119] As used herein, and unless otherwise specified, the phrase "hierarchical vascular network" refers to a vascular network within the three-dimensional biological cell structure that comprises vessels of varying sizes. For example, it may comprise arterioles, capillaries, and venules. The arterioles may transition to capillaries, which then transition to venules. It typically exhibits an organized arrangement similar to natural vascular systems, enabling efficient perfusion and more closely replicating in vivo tissue characteristics.

[0120] As used herein, and unless otherwise specified, the phrase "anastomosed vasculature formed by angiogenesis and / or vasculogenesis from a hydrogel compartment into the three-dimensional biological cell structure" refers to a vascular network where endothelial cells have formed interconnected vessels that extend from a hydrogel compartment into the three-dimensional biological cell structure, creating functional connections through processes of new vessel formation and network development.

[0121] As used herein, and unless otherwise specified, the term "flow controller" refers to a device or system capable of regulating fluid flow parameters such as rate, pressure, or timing within the microfluidic channels of the microfluidic device. It typically facilitates controlled delivery or removal of culture media, cells, or other substances to support cell culture and experimental protocols.As used herein, and unless otherwise specified, the phrase "controlled flow of culture medium" refers to the deliberate and regulated movement of culture medium through the pair of microfluidic channels. Typically, parameters like flow rate and pressure are adjusted. This enables maintaining good conditions for cell survival, growth, and vascular network development.

[0122] As used herein, and unless otherwise specified, the phrase "introducing endothelial cells and pericytes" refers to the process of adding these specific types of cells into a hydrogel compartment or microfluidic channels. Endothelial cells form the lining of vascular structures and pericytes contribute to vessel stability and maturation, facilitating the formation of a functional vascular network.

[0123] As used herein, and unless otherwise specified, the phrase "harvesting the vascularized three-dimensional biological cell structures" refers to removing the cultured three-dimensional biological cell structures, which have developed a vascular network, from the microfluidic device. This can be for purposes such as analysis, further culture, transplantation, or other applications.

[0124] As used herein, and unless otherwise specified, the phrase "assess effects on the three-dimensional biological cell structure" refers to evaluating how the three-dimensional biological cell structures respond to experimental conditions, treatments, or compounds introduced into the culture medium, including assessment of cell viability, function, morphology, gene expression, or other relevant biological parameters.

[0125] The invention will now be described by a detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be configured according to the knowledge of persons skilled in the art without departing from the technical teaching of the invention, the invention being limited only by the terms of the appended claims.

[0126] In a first aspect, the present invention relates to a microfluidic device (100) for culturing three-dimensional biological cell structures (35).

[0127] As illustrated in Figure 1, which shows a detailed cross-sectional view of a docking site (36) of a microfluidic device (100) according to embodiments of the present invention, the device (100) comprises a first level (10) with a plurality of microfluidic channels (11), including a pair of microfluidic channels (11). A second level (20), different from the first level (10), comprises one or more hydrogel compartments (21) (one is depicted) for hosting a hydrogel (22). Each hydrogel compartment (21) is in fluid communication with the pair of microfluidic channels (11). A substrate (31) having a top surface (32) forms a wall (33) of acell culture compartment (34) for hosting one or more three-dimensional biological cell structures (35). The culture compartment (34) includes one or more three-dimensional biological cell structure docking sites (36), each comprising a through-hole (37) with a top surface (38) having an area. The through-hole (37) ensures fluid communication between the cell culture compartment (34) and a hydrogel compartment (21) and is situated laterally between the pair of microfluidic channels (11). A docking structure (39) is provided for docking a three-dimensional biological cell structure (35) so that it is in fluidic communication with the through-hole (37).

[0128] In embodiments, the second level (20) may be disposed above the first level (10). This configuration allows for efficient stacking of the microfluidic channels (11) and hydrogel compartments (21).

[0129] In embodiments, the substrate (31) may be comprised in a third level (30), disposed above the second level (20), wherein the substrate (31) forms the lowest part of the third level (30) and a bottom wall (33) of the cell culture compartment (34). This three-level architecture enables optimal spatial arrangement of the components.

[0130] In embodiments, the docking structure (39) may be designed for docking the three-dimensional biological cell structure (35) so that it covers at least partly (e.g., completely) the through-hole (37). This ensures proper fluidic communication between the three-dimensional biological cell structure (35) and the hydrogel compartment (21).

[0131] Referring to Figure 2, which depicts a cross-sectional view of a docking site (36) of a microfluidic device (100) with a three-dimensional biological cell structure (35) docked thereto according to embodiments of the present invention, the three-dimensional biological cell structure (35) is shown docked to the docking structure (39) and covering the through-hole (37). This arrangement allows fluidic communication between the three-dimensional biological cell structure (35), a hydrogel compartment (21), and the pair of microfluidic channels (11).

[0132] In embodiments, the three-dimensional biological cell structure (35) may be a type of biological cell structure that requires vascularization to survive. The device (100) is particularly suited for culturing cell structures with high vascularization needs.

[0133] In embodiments, the three-dimensional biological cell structure (35) may be selected from organoids, spheroids, and three-dimensional tissue engineering constructs. These are examples of relevant biological structures that can be cultured in the device (100).

[0134] In embodiments, the docking structure (39) is configured such that, if one closes its uppermost boundary by a horizontal plane, one or more openings are comprised in itsoutermost lateral boundary. This configuration facilitates lateral access to the docked biological cell structure (35).

[0135] In embodiments, the innermost lateral boundary of the docking structure (39) may be at all points laterally separated from the top surface (38) of the through-hole (37) by a distance of at most 10%, preferably at most 5%, yet more preferably at most 2% of the largest lateral dimension of the top surface (38) of the through-hole (37). Limiting this separation distance ensures compact docking.

[0136] In embodiments, the docking structure (39) may comprise one or more protrusions (41) projecting from the top surface (32) of the substrate (31). The protrusions (41) aid in securely docking the three-dimensional biological cell structure (35).

[0137] In embodiments, the one or more protrusions (41) may be projecting vertically from the top surface (32) of the substrate (31). Vertical protrusions (41) provide optimal structural support.

[0138] In embodiments, the one or more protrusions (41) may be a plurality of spaced apart protrusions (41). Spaced apart protrusions (41) allow for fluid access between them.

[0139] In embodiments, the one or more protrusions (41) may be a plurality of protrusions (41) in a number of at least 3, preferably from 3 to 10, more preferably from 4 to 8, yet more preferably from 5 to 7, such as 6. This range of protrusion numbers has been found to provide good docking performance.

[0140] In embodiments, the one or more protrusions (41) may be regularly spaced. Regular spacing provides a uniform docking interface.

[0141] In embodiments, the plurality of spaced apart protrusions (41) may be surrounding the through-hole (37). Surrounding the through-hole (37) with protrusions (41) facilitates centering the three-dimensional biological cell structure (35) over it.

[0142] In embodiments, all protrusions (41) may be at a same distance from the through-hole (37). Equidistant protrusions (41) provide balanced structural support.

[0143] In embodiments, the protrusions (41) may be pillars. Pillars are simple yet effective protrusion structures.

[0144] In embodiments, the largest distance between any two neighboring protrusions (41) may be from 100 to 200 pm. This distance range allows adequate biological cell structure (35) support while permitting fluid access.

[0145] Referring to Figure 4, which presents a top view of a docking site (36) of a microfluidic device (100) according to embodiments of the present invention, the docking structures (39) are shown surrounding the through-holes (37). As shown in Figure 5, the pairof microfluidic channels (11) is situated laterally on either side of the through-hole (37). The arrangement of the protrusions (41) can be clearly seen, facilitating the docking of the three-dimensional biological cell structure (35) over the through-hole (37).

[0146] In embodiments, the pair of microfluidic channels (11) may comprise an inlet channel (43) for bringing matter into the hydrogel (22) and an outlet channel (44) for removing matter from the hydrogel (22). Dedicated inlet and outlet channels enable efficient fluid exchange.

[0147] In embodiments, the pair of microfluidic channels (11) may be arranged in parallel. A parallel arrangement provides a streamlined fluid circuit.

[0148] In embodiments, the device (100) may comprise at least two docking sites (36), preferably from 10 to 20 docking sites (36), situated between said pair of microfluidic channels (11). Multiple docking sites (36) allow simultaneous culturing of several three-dimensional biological cell structures (35).

[0149] As illustrated in Figure 5, which depicts a top view of a microfluidic device (100) comprising a plurality of docking sites (36) according to embodiments of the present invention, the device (100) includes multiple pairs of microfluidic channels (11), with docking sites (36) situated laterally between each pair. In this embodiment, dashed-line rectangles (21) are shown as individual hydrogel compartments in fluid communication with each of the multiple pairs of microfluidic channels (11). This arrangement allows for the culturing and vascularization of multiple three-dimensional biological cell structures (35) in parallel, enabling high-throughput experimentation and analysis.

[0150] In embodiments, these at least two docking sites (36) may be distributed amongst a plurality of hydrogel compartments (21). For instance, the number of docking sites (36) situated between said pair may be equal to the number of hydrogel compartments (21). This corresponds to a situation where each hydrogel compartment (21) portion present between the pair of microfluidic channels (11) has exactly one docking site (36) with its associated through-hole (37). This is the situation depicted in figure 5. As another example, the number of docking sites (36) situated between said pair may be equal to an integer multiple of the number of hydrogel compartment (21). This corresponds to a situation where each hydrogel compartment (21) portion present between the pair of microfluidic channels (11) has multiple docking sites (36), for example two, three, or four docking sites (36), each with its associated through-hole (37). In such a situation, biological three-dimensional biological cell structure (35) of a same type may occupy each of the multiple docking sites (36) or differentbiological three-dimensional biological cell structure (35) may be present at the multiple docking sites (36).

[0151] In embodiments, the microfluidic channels (11) of the pair may be parallel and said at least two docking sites (36) may have their through-holes (37) aligned in a first direction parallel to the direction of the microfluidic channels (11) of the pair. Aligned through-holes (37) facilitate uniform fluid delivery to the three-dimensional biological cell structures (35).

[0152] In embodiments, the plurality of microfluidic channels (11) may comprise at least three microfluidic channels (11) forming a first and a second pair of microfluidic channels (11) sharing a central microfluidic channel, a first and a second series of docking sites (36), wherein said first series of docking sites (36) is laterally between the microfluidic channels (11) of the first pair, and wherein said second series of docking sites (36) is laterally between the microfluidic channels (11) of the second pair. This configuration enables high-density docking site packing.

[0153] In embodiments, the first and second series of docking sites (36) may have their through-holes (37) aligned in the first direction. Alignment in the flow direction promotes uniform fluid access.

[0154] As further illustrated in Figure 3, which shows a perspective view of a microfluidic device (100) with a plurality of three-dimensional biological cell structures (35) docked thereto according to embodiments of the present invention, multiple docking sites (36) are shown, each with a three-dimensional biological cell structure (35) docked. This demonstrates the capability of the device (100) to host and culture multiple three-dimensional biological cell structures (35) simultaneously.

[0155] In embodiments, each hydrogel compartment (21) may be oblong and have its longitudinal direction forming an angle, preferably a 90° angle, with the longitudinal direction of each microfluidic channel (11) of the pair.

[0156] In embodiments, each hydrogel compartment (21) may be in fluid communication with said pair of microfluidic channels (11) via openings (50) having a hydraulic diameter of from 5 to 200 micrometers, preferably 20 to 150 micrometers, more preferably 60 to 130 micrometers. These opening size ranges allow proper hydrogel retention while permitting fluid exchange.

[0157] In embodiments, the docking structure (39) may have an outermost boundary having a maximal length measured parallel to the top surface (32) of the substrate (31) of from 50 to 400 micrometers, preferably from 70 to 300 micrometers, more preferably from 80 to 250 pm. These dimensions are well-suited for typical biological cell structure sizes.In embodiments, the microfluidic channels (11) may have a height (h) of from 50 to 300 micrometers. This height range provides a good balance between fluid flow and device compactness.

[0158] In embodiments, the microfluidic channels (11) may have a width (w) of from 50 to 300 micrometers. This width range accommodates typical fluid flow needs.

[0159] In embodiments, the device (100) may be at least partly fabricated from optically transparent materials so that when three-dimensional biological cell structures (35) are present, they can be observed from the top and / or the bottom of the device (100). Optical transparency enables monitoring of the three-dimensional biological cell structures (35). It also allows qualitative and quantitative characterization of flow patterns and vascular network formation, facilitating real-time observation and analysis.

[0160] In embodiments, the device (100) may be at least partially fabricated from materials having a UV light transmission of at least 80% so as to enable UV-initiated crosslinking of the hydrogel (22) when loaded in the hydrogel compartment (21).

[0161] In embodiments, the device (100) may be at least partially fabricated from materials selected from optically transparent polymers having a thickness and UV transmission properties that ensure UV light attenuation does not exceed 20% during hydrogel (22) crosslinking processes. Examples of such materials include poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PM MA), and cyclic olefin copolymer (COC).

[0162] In embodiments, the device (100) may further comprise a biosensor (51) configured for measuring at least one parameter from a three-dimensional biological cell structure (35) when it is docked at a docking site (36) and / or configured for detecting a substance in the cell culture compartment (34). Integrated biosensors (51) allow real-time monitoring of the biological environment.

[0163] In embodiments, said biosensor (51) may comprise one or more electrodes.

[0164] Electrodes are versatile sensors for biological systems.

[0165] In embodiments, the device (100) may further comprise the hydrogel (22) and the three-dimensional biological cell structures (35).

[0166] In embodiments, the three-dimensional biological cell structures (35) may each be of a type that requires vascularization to survive. The device (100) is particularly suited for culturing cell structures with high vascularization needs.

[0167] In embodiments, each of the three-dimensional biological cell structures (35) may be independently selected from organoids, spheroids, and three-dimensional tissue engineering constructs. These are non-limiting examples of relevant biological structures that can becultured in the device (100). Preferably, each of the three-dimensional biological cell structure (35) is an organoid.

[0168] In embodiments, the three-dimensional biological cell structures (35) may comprise different three-dimensional biological cell structures (35).

[0169] In embodiments, the three-dimensional biological cell structures (35) may each be a human organoid independently selected from the group consisting of brain organoids, liver organoids, kidney organoids, cardiac organoids, gut organoids, and tumor organoids. These organoid types are of great biomedical interest, but the present invention is applicable to any organoids.

[0170] In embodiments, the three-dimensional biological cell structures (35) docked on docking sites (36) comprising a through-hole (37) ensuring fluid communication between the cell culture compartment (34) and a same hydrogel compartment (21), comprise different three-dimensional biological structures (35). In other words, the three-dimensional biological cell structures (35) in fluid communication with the same hydrogel compartment (21) may comprise different three-dimensional biological structures (35). For instance, the three-dimensional biological cell structures (35) in fluid communication with the same hydrogel compartment (21) may comprise different human organoids, for instance selected from the group consisting of brain organoids, liver organoids, kidney organoids, cardiac organoids and gut organoids. In embodiments, several or all of these different human organoids (brain organoids, liver organoids, kidney organoids, cardiac organoids, and gut organoids) may be present, thereby forming a "human-on-chip."

[0171] In embodiments, the three-dimensional biological cell structures (35) may be vascularized. Vascularization helps long-term cell structure viability.

[0172] In embodiments, the device (100) may further comprise a vascular network within the hydrogel (22), said vascular network being vascularly connected to the three-dimensional biological cell structures (35). An integrated vascular network supplying the three-dimensional biological cell structures (35) greatly enhances their growth and survival.

[0173] In embodiments, the vascular network may comprise anastomosed vasculature formed by angiogenesis and / or vasculogenesis from a hydrogel compartment (21) into the three-dimensional biological cell structure (35). Angiogenesis and vasculogenesis are effective natural mechanisms for generating vascular networks.

[0174] In second aspect, the present invention relates to a system (200) comprising a device (100) according to any one of the preceding claims, and a flow controller (55) for applying controlled flow of culture medium through a microfluidic channel (11) of the pair. A flowcontrol system allows optimizing the fluid environment for the three-dimensional biological cell structures (35). This is depicted in Figure 1.

[0175] In a third aspect, the present invention relates to a method of vascularizing three-dimensional biological cell structures (35), comprising providing a system (200) according to any embodiment of the present invention, introducing endothelial cells and pericytes into the one or more hydrogel compartments (21), providing three-dimensional biological cell structures (35) at the docking sites (36), and applying controlled flow of culture medium through the pair of microfluidic channels (11), thereby vascularizing the three-dimensional biological cell structures (35).

[0176] In embodiments, the method may further comprise introducing endothelial cells and / or pericytes into the microfluidic channels (11) to endothelialize the channels. In embodiments, other vascular-supporting cells may also be introduced into the microfluidic channels (11). Endothelialized channels provide a more physiological fluid delivery system.

[0177] In embodiments, the three-dimensional biological cell structures (35) may be introduced as pre-formed three-dimensional biological cell structures (35). Pre-forming the cell structures (35) allows optimizing their initial composition and organization.

[0178] In embodiments, the three-dimensional biological cell structures (35) may be formed in situ by introducing cell suspensions in contact with the docking sites (36). In situ formation simplifies the cell seeding process.

[0179] In embodiments, the method may further comprise harvesting the vascularized three-dimensional biological cell structures (35). Harvesting allows detailed analysis and further use of the cell structures (35).

[0180] In embodiments, multiple types of three-dimensional biological cell structures (35) may be cultured simultaneously at different docking sites (36). Simultaneous culturing of different cell structure types allows studying their interactions.

[0181] In embodiments, the endothelial cells and pericytes may be derived from human cells, e.g., from human primary cells or human induced pluripotent stem cells. Using human cells makes the method more clinically relevant.

[0182] In embodiments, the three-dimensional biological cell structures (35) may comprise human organoids selected from the group consisting of brain organoids, liver organoids, kidney organoids, gut organoids, cardiac organoids, and tumor organoids. These organoid types are of great biomedical interest.

[0183] In embodiments, the microfluidic channels (11) and the hydrogel compartments (21) may be arranged at an angle, and may preferably be arranged perpendicularly. This facilitatesa human-on-chip application. In this configuration, different organoid types can be docked at different docking sites (36) in fluidic communication with the same hydrogel compartment (21), with all organoids being interconnected through a common vascular network developing within the hydrogel (22).

[0184] This design architecture of the device (100), where the microfluidic channels (11) intersect the hydrogel compartment (21) at an angle (e.g. of from 80 to 100° such as 90°), facilitates the creation of an integrated multi-organ system. The different three-dimensional biological cell structures (35) can communicate through the shared vascular network while maintaining their individual positions at their respective docking sites (36), thereby enabling the study of organ-organ interactions and systemic responses in a controlled microenvironment.

[0185] In embodiments, applying controlled flow may comprise applying a pressure difference between the first and second microfluidic channel (11) of the pair to generate pressure gradients within the pair. Pressure gradients can be used to mimic in vivo interstitial flow.

[0186] In embodiments, the method may further comprise introducing compounds or pathogens into the culture medium to assess effects on the three-dimensional biological cell structure (35). The platform is well-suited for drug testing and screening. The method is also well-suited for the determination of pathogen biodistribution and infection routes.

[0187] In fourth aspect, the present invention relates to a vascularized biological cell structure (35) produced by the above method. The vascularized cell structures (35) have wide applications in research and therapeutics.

[0188] In embodiments, the three-dimensional biological cell structure (35) may comprise a hierarchical vascular network. A hierarchical vascular network improves cell structure (35) perfusion and function.

[0189] It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope of this invention. For example, any formulas given above are merely representative of procedures that may be used. Functionality may be added or deleted from the block diagrams and operations may be interchanged among functional blocks. Steps may be added or deleted to methods described within the scope of the present invention.

Claims

1. 26Claims1. A microfluidic device (100) for culturing three-dimensional biological cell structures (35), comprising:- a first level (10) comprising a plurality of microfluidic channels (11) comprising a pair of microfluidic channels (11);- a second level (20), different from the first level (10), comprising one or more hydrogel compartments (21) for hosting a hydrogel (22), wherein each hydrogel compartment (21) is in fluid communication with said pair of microfluidic channels (11); and- a substrate (31) having a top surface (32), said substrate (31) forming:- a wall (33) of a cell culture compartment (34) for hosting one or more three-dimensional biological cell structures (35), said cell culture compartment (34) comprising one or more biological cell structure docking sites (36), each docking site (36) comprising:a through-hole (37) having a top surface (38) having an area, said through-hole (37) ensuring fluid communication between the cell culture compartment (34) and a hydrogel compartment (21), said through- hole (37) being situated laterally between the pair of microfluidic channels (11); anda docking structure (39) for docking a three-dimensional biological cell structure (35) so that the three-dimensional biological cell structure (35) is in fluidic communication with the through-hole (37).

2. The microfluidic device (100) according to claim 1, wherein the substrate is comprised in a third level (30), disposed above the second level (20), wherein the substrate forms the lowest part of the third level (30) and a bottom wall (33) of the cell culture compartment (34).

3. The microfluidic device (100) according to claim 1 or claim 2 wherein the docking structure (39) is for docking the three-dimensional biological cell structure (35) so that the three-dimensional biological cell structure (35) covers at least partly and preferably completely the through-hole (37).

4. The microfluidic device (100) according to any of the preceding claims, wherein the docking structure (39) comprises one or more protrusions (41) projecting from the top surface (32) of the substrate (31).

5. The microfluidic device (100) according to any one of the preceding claims, wherein the pair of microfluidic channels (11) comprises an inlet channel (43) for bringing matter into the hydrogel (22) and an outlet channel (44) for removing matter from the hydrogel (22).

6. The microfluidic device (100) according to any one of the preceding claims, comprising at least two docking sites (36), preferably from 10 to 20 docking sites (36), situated between said pair of microfluidic channels (11).

7. The microfluidic device (100) according to any one of the preceding claims, wherein the pair of microfluidic channels (11) is arranged in parallel.

8. The microfluidic device (100) according to claim 7, wherein the plurality of microfluidic channels (11) comprises:at least three microfluidic channels (11) forming a first and a second pair of microfluidic channels (11) sharing a central microfluidic channel,a first and a second series of docking sites (36), wherein said first series of docking sites (36) is laterally between the microfluidic channels (11) of the first pair, and wherein said second series of docking sites (36) is laterally between the microfluidic channels (11) of the second pair.

9. The microfluidic device (100) according to claim 7 or claim 8, wherein each hydrogel compartment (21) is oblong and has its longitudinal direction forming an angle,preferably a 90° angle, with the longitudinal direction of each microfluidic channel (11) of the pair.

10. The microfluidic device (100) according to any one of the preceding claims, wherein each hydrogel compartment (21) is in fluid communication with said pair of microfluidic channels (11) via openings (50) having a hydraulic diameter of from 5 to 200 micrometers, preferably 20 to 150 micrometers, more preferably 60 to 130 micrometers.

11. The microfluidic device (100) according to any one of the preceding claims, further comprising a biosensor (51) configured for measuring at least one parameter of a three-dimensional biological cell structure (35) when it is docked at a docking site (36) and / or configured for detecting a substance in the cell culture compartment (34).

12. The microfluidic device (100) according to any one of the preceding claims, further comprising the hydrogel (22) and the three-dimensional biological cell structures (35).

13. A system (200) comprising a device (100) according to any one of the preceding claims, and a flow controller (55) for applying controlled flow of culture medium through a microfluidic channel (11) of the pair.

14. A method of vascularizing three-dimensional biological cell structures (35), comprising:- providing a system (200) according to claim 13;- introducing endothelial cells and pericytes into the hydrogel compartments (21);- providing three-dimensional biological cell structures (35) at the docking sites (36);- applying controlled flow of culture medium through the pair of microfluidic channels (11), thereby vascularizing the three-dimensional biological cell structures (35).2915. A vascularized three-dimensional biological cell structure (35) produced by the method of claim 14.

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