Methods and systems for in vitro formation and culture of a vascularized three-dimensional tissue model

Abstract

The present disclosure relates to a method and system for the in vitro formation and culture of a vascularized three-dimensional tissue model, for initiating culturing and / or differentiation of cells and, simultaneously, injecting at least one second fluid (13) into at least one chamber (100) containing a first fluid (17), for establishing a laminar flow of said at least one second fluid (13) between at least a first fluid inlet (10) and at least a first fluid outlet (11), then initiating solidification of said first fluid (17) to obtain a gel (19), while maintaining said laminar flow to prevent the formation of said gel (19) and create at least one channel. The method and the system according to the invention make it possible to maintain the culturing and / or differentiation of the cells until notably said vascularized three-dimensional tissue model is obtained.

Description

[0001] TITLE: METHODS AND SYSTEMS FOR IN VITRO FORMATION AND CULTURE OF A THREE-DIMENSIONAL VASCULARIZED TISSUE MODEL.

[0002] DESCRIPTION

[0003] TECHNICAL FIELD

[0004] This disclosure relates to the in vitro culture of three-dimensional cellular tissues. More specifically, this disclosure relates to the active vascularization of such three-dimensional cellular tissues in vitro. It should be noted that the term "active vascularization" refers to the ability to dynamically generate, maintain, and adapt a network of blood vessels supplying three-dimensional cellular tissues in vitro, similar to an in vivo vascular network.

[0005] STATE OF PRIOR ART

[0006] Three-dimensional, or 3D, cell culture makes it possible to mimic the organization and microarchitecture of living organs or tissues. Currently, this technology is used in areas such as the development of therapies, disease research, and tissue regeneration research, in order to bridge the gap between in vitro and in vivo models.

[0007] However, limitations remain when it comes to creating in vitro 3D tissue models with sufficient and realistic vascular supply. Vascularization of existing 3D tissue models is essential to mimic the physiological and pathophysiological conditions of the human body and its tissues, reproduce the physiological connections between different tissue types in vitro, and maintain these tissues viable for extended periods. Furthermore, fabricating in vitro 3D tissue models irrigated by a vascular network perfused by systems that monitor and control the flow and composition of fluids suitable for the in vitro formation and culture of such 3D tissue models also presents a challenge.

[0008] Currently, to address the problem of vascularizing 3D tissues, in vitro vascular networks are generated, for example, using pre-formed hollow channel systems. These pre-formed channels are lined with vascular cells to generate vascular-like networks. However, constraining the cells of the vascular system to a specific predefined geometry limits the ability of these vascular-like networks to reorganize and grow in response to stimuli. This approach offers limited temporal and spatial control over the simultaneous development of vascular and parenchymal tissue structures, as the vessel architecture is pre-formed during the manufacturing process.Furthermore, any parenchymal tissue formation is limited to the residual extravascular space, and there is no mutual biochemical exchange during the initial stages of tissue formation, which are important for the phenotypes and / or differentiation of the constituent cells. For example, in nature, the formation of organotypic vascular networks requires interaction between differentiating parenchymal cells and developing blood vessels.

[0009] In another example, microvascular networks can be formed using the self-assembly capacity of vascular cells. For this to occur, the vascular cells grow and self-organize within a preformed gel exhibiting certain characteristics of the extracellular matrix and / or other parenchymal cells. However, this approach also has limitations.

[0010] It is therefore desirable to overcome these drawbacks of the state of the art.

[0011] It is particularly desirable to provide a much more versatile and physiological solution for the in vitro culture of three-dimensional tissue models, allowing for temporal and spatial control of the simultaneous development of vascular and parenchymal tissue structures. Furthermore, it is essential that the in vitro vascularized parenchymal tissues be properly and uniformly connected to the vascular network and actively perfused in a controlled manner, generating the expected and necessary biophysical and biomolecular signaling interactions, while also being able to reach the depths of the various parts of the generated parenchymal structures.

[0012] DESCRIPTION OF THE INVENTION

[0013] A method for the in vitro formation and culture of a three-dimensional vascularized tissue model is proposed herein. The method comprises: introducing into at least one chamber a first fluid comprising gel precursors intended to form one or more three-dimensional networks under the influence of a predetermined factor or a combination of predetermined factors, said first fluid further comprising cells belonging to at least one first cell group comprising cells belonging to one or more cell types of mature and / or stem cells associated with the parenchyma of one or more target tissues to be imitated or replicated, and / or to at least one second cell group comprising cells belonging to one or more cell types of mature and / or stem cells associated with the stroma. The method is characterized in that it further comprises:

[0014] - to initiate the culture and / or differentiation of said cells of said at least one first cell group and / or at least one second cell group, and, simultaneously, - to inject at least one second fluid into said at least one chamber containing said first fluid, by at least one first fluid control device, through at least one first fluid inlet, said at least one first fluid inlet being immersed in said first fluid, and said at least one second fluid flowing from said at least one first fluid inlet to at least one first fluid outlet immersed in said first fluid, to create at least one flow jet and establish a laminar flow of said at least one second fluid between said at least one first fluid inlet and said at least one first fluid outlet,

[0015] - then, initiate a solidification of said first fluid by modifying said predetermined factor or at least one of said predetermined factors of said combination to obtain a gel, while maintaining said laminar flow to prevent the formation of said gel and create at least one channel between said at least one first fluid inlet and said at least one first fluid outlet,

[0016] - maintain the culture and / or differentiation of cells belonging to said at least one second cell group included in said first fluid and / or in said at least one second fluid until cells belonging to said at least one second group totally or partially colonize at least one wall of said at least one channel, - maintain the culture and / or differentiation of said cells belonging to said at least one first cell group and to said at least one second cell group until said three-dimensional vascularized tissue model is obtained.

[0017] It is thus possible to create a three-dimensional tissue model (e.g., cells, tumoroid organoids, embryoids, assembloids, etc.) vascularized and connected to one or more fluid control devices that allow perfusion of the resulting 3D tissue model. In particular, it is possible to generate, in vitro, in three dimensions, the stroma and parenchyma of tissue models of interest by exploiting and controlling the dynamics of fluids perfused simultaneously with the formation of a gel and the generation and maturation of tissues embedded in the gel and / or in the perfused fluids.

[0018] To this end, the process according to the invention makes it possible to create channels (or cavities or fluidic passages) by preventing the formation of a gel along the path of a laminar flow generated by the flow of a fluid within the gel being formed. Concurrently with the formation of the gel and the channels traversing it, the cells embedded in the fluid intended to solidify to obtain the gel and / or in the injected fluid are free to organize themselves and generate cell assemblies, stromal structures, organoids, assembloids, tumoroids, embryoids, and / or biological tissues around these channels.

[0019] Furthermore, perfusion during the genesis and maturation of these in vitro 3D tissue models, through the creation of these channels, allows them to be exposed to one or more agents (e.g., oxygen, biomolecules), or to gradients of agents, and to physical forces (e.g., pressure, shear stress) in a more physiological or pathophysiological manner. This makes it possible to obtain physiologically relevant perfused parenchyma models, particularly in complex biological models such as tissues of the central nervous system, the respiratory system, or the digestive system, as well as tumor tissues.

[0020] According to a particular embodiment, cells belonging to said at least a second cell group are included at least in said at least a second fluid for a predetermined period before and / or during and / or after said solidification of said first fluid.

[0021] According to a particular embodiment, the process further comprises: adjusting over time a pressure exerted on the top of said first fluid before and / or during and / or after said solidification of said first fluid.

[0022] According to a particular embodiment, the process further comprises: introducing at least one third fluid into said at least one chamber, before and / or during and / or after said solidification of said first fluid, said third fluid comprising one or more or a combination of the following elements:

[0023] (i) cells belonging to said at least one first cell group;

[0024] (ii) cells belonging to one or more other cell types; (iii) compounds intended to produce effects on some or all of the cells of said at least one first cell group and / or on some or all of the cells of said at least one second cell group and / or on some or all of the cells belonging to one or more other cell types;

[0025] (iv) chemical or biochemical agents capable of initiating said solidification of said first fluid and corresponding to said predetermined factor or at least to one of said predetermined factors of said combination;

[0026] (v) a cell culture and / or cell differentiation medium suitable for the culture and / or differentiation of cells of said at least one first cell group and / or of said at least one second cell group and / or of said or other cell types; (vi) a gas or mixture of gases or a liquid comprising a predetermined amount of one or more dissolved gases.

[0027] According to a particular embodiment, the process further comprises: modifying a composition of said at least one second fluid or changing said at least one second fluid by at least one other second fluid for a predetermined period, after said solidification of said first fluid, to introduce and / or remove from said at least one second fluid one or both or a combination of the following:

[0028] (i) cells belonging to said at least a second cell group;

[0029] (ii) cells belonging to one or more other cell types;

[0030] (iii) compounds intended to produce effects on some or all of the cells of said at least one first cell group and / or on some or all of the cells of said at least one second cell group and / or on some or all of the cells belonging to one or more other cell types;

[0031] (iv) chemical or biochemical agents capable of initiating said solidification of said first fluid and corresponding to said predetermined factor or at least to one of said predetermined factors of said combination;

[0032] (vi) liquids comprising predetermined amounts of gas or mixture of dissolved gases to produce at least one dissolved gas gradient in the gel.

[0033] According to a particular embodiment, said predetermined factor or at least one of said predetermined factors of said combination is: a temperature or a range of temperatures, the solidification of said first fluid being initiated by modifying the initial temperature of said first fluid and / or of said at least a second fluid to reach a predetermined temperature or temperature gradient at which the first fluid gradually solidifies to obtain the gel.

[0034] According to a particular embodiment, the process further comprises: adjusting over time a flow rate of said at least one second fluid generated by said at least one fluid control device.

[0035] According to a particular embodiment, the process further comprises: adjusting over time a temperature of said at least a second fluid.

[0036] Also proposed here is an in vitro training and culture system for a three-dimensional vascularized tissue model. The system is configured to: introduce into at least one chamber a first fluid comprising gel precursors intended to form one or more three-dimensional networks under the influence of a predetermined factor or a combination of predetermined factors, said first fluid further comprising cells belonging to at least a first cell group comprising cells belonging to one or more cell types of mature and / or stem cells associated with the parenchyma of one or more target tissues to be imitated or replicated, and / or at least a second cell group comprising cells belonging to one or more cell types of mature and / or stem cells associated with the stroma.The system is further configured to: - initiate culture and / or differentiation of said cells of said at least one first cell group and / or at least one second cell group, and, simultaneously, - inject at least one second fluid into said at least one chamber containing said first fluid, by at least one first fluid control device, through at least one first fluid inlet, said at least one first fluid inlet being immersed in said first fluid, and said at least one second fluid flowing from said at least one first fluid inlet to at least one first fluid outlet immersed in said first fluid, to create at least one flow jet and establish a laminar flow of said at least one second fluid between said at least one first fluid inlet and said at least one first fluid outlet.

[0037] - then, initiate a solidification of said first fluid by modifying said predetermined factor or at least one of said predetermined factors of said combination to obtain a gel, while maintaining said laminar flow to prevent the formation of said gel and create at least one channel between said at least one first fluid inlet and said at least one first fluid outlet,

[0038] - maintain the culture and / or differentiation of cells belonging to said at least one second cell group included in said first fluid and / or in said at least one second fluid until cells belonging to said at least one second group totally or partially colonize at least one wall of said at least one channel, - maintain the culture and / or differentiation of said cells belonging to said at least one first cell group and to said at least one second cell group until said three-dimensional vascularized tissue model is obtained.

[0039] According to a particular embodiment, the system is configured to: adjust over time a pressure exerted on the top of said first fluid before and / or during and / or after said solidification of said first fluid.

[0040] According to a particular embodiment, the system is configured to: introduce at least one third fluid into said at least one chamber, before and / or during and / or after said solidification of said first fluid, said third fluid comprising one or more or a combination of the following elements:

[0041] (i) cells belonging to said at least one first cell group;

[0042] (ii) cells belonging to one or more other cell types;

[0043] (iii) compounds intended to produce effects on some or all of the cells of said at least one first cell group and / or on some or all of the cells of said at least one second cell group and / or on some or all of the cells belonging to one or more other cell types;

[0044] (iv) chemical or biochemical agents capable of initiating said solidification of said first fluid and corresponding to said predetermined factor or at least to one of said predetermined factors of said combination;

[0045] (v) a cell culture and / or cell differentiation medium suitable for culturing and / or differentiating cells of said at least one first cell group and / or said at least one second cell group and / or said or said other cell types; (vi) a gas or mixture of gases or a liquid comprising a predetermined amount of one or more dissolved gases.

[0046] According to a particular embodiment, the system is further configured to: modify the composition of said at least one second fluid or change said at least one second fluid by at least one other second fluid during a predetermined period, after said solidification of said first fluid, to introduce and / or remove from said at least one second fluid one or both or a combination of the following:

[0047] (i) cells belonging to said at least a second cell group;

[0048] (ii) cells belonging to one or more other cell types;

[0049] (iii) compounds intended to produce effects on some or all of the cells of said at least one first cell group and / or on some or all of the cells of said at least one second cell group and / or on some or all of the cells belonging to one or more other cell types;

[0050] (iv) chemical or biochemical agents capable of initiating said solidification of said first fluid (17) and corresponding to said predetermined factor or at least to one of said predetermined factors of said combination;

[0051] (vi) liquids comprising predetermined amounts of gas or mixture of dissolved gases to produce at least one dissolved gas gradient in the gel.

[0052] According to a particular embodiment, the system is further configured to: adjust over time a flow rate of said at least one second fluid generated by said at least one fluid control device.

[0053] According to a particular embodiment, the system is further configured to: adjust over time a temperature of said at least a second fluid.

[0054] Thus, the invention also relates to a system for the in vitro formation and culture of a three-dimensional vascularized tissue model, said system comprising:

[0055] - a chamber capable of containing a fluid, said chamber comprising at least one first fluid inlet and at least one first fluid outlet; and

[0056] - at least one fluid control device connected to said at least one first inlet; characterized in that said system is configured such that, when the chamber contains a first fluid comprising gel precursors in the liquid state or in transition from a liquid to a solid state, said fluid control device is capable of injecting at least one second fluid to form at least one flow jet and establish at least one laminar flow from said inlet to said outlet within said first fluid. According to a particular embodiment, said system further comprises at least one second fluid inlet connected to the chamber and capable of introducing at least one third fluid into said chamber.

[0057] According to certain particular embodiments of the systems according to the invention, the fluid control device is further configured to adjust over time a pressure exerted on said first fluid.

[0058] According to certain particular embodiments of the systems according to the invention, the fluid control device is configured to selectively inject into the chamber one of a plurality of second fluids having different compositions.

[0059] According to a particular embodiment, said system further comprises at least one temperature control device configured to modify the temperature of said first fluid in the chamber in order to initiate its solidification.

[0060] According to certain particular embodiments of the systems according to the invention, the fluid control device is configured to adjust the flow rate of at least one of said second and third fluids.

[0061] According to one particular embodiment, said system further comprises at least one temperature control device configured to adjust the temperature of at least one second fluid. According to particular embodiments applicable to all in vitro methods for forming and culturing a three-dimensional vascularized tissue model and to all in vitro systems for forming and culturing a three-dimensional vascularized tissue model according to the invention, said stem cells are not human embryonic stem cells. BRIEF DESCRIPTION OF DRAWINGS

[0062] The features of the invention mentioned above, as well as others, will become clearer upon reading the following description of at least one exemplary embodiment, said description being made in relation to the accompanying drawings, among which:

[0063] [Fig. IA], [Fig. IB], [Fig. IC], [Fig. 1D] schematically illustrate an implementation of a process for the formation and in vitro culture of a three-dimensional vascularized tissue model, according to particular embodiments;

[0064] [Fig. 2A], [Fig. 2B], [Fig. 2C], [Fig. 2D] schematically illustrate examples of the arrangement of one or more cell culture chambers adapted to implement a process of in vitro formation and culture of a three-dimensional vascularized tissue model, according to particular embodiments;

[0065] [Fig. 3A], [Fig. 3B], [Fig. 3C] schematically illustrate examples of laminar flows generated by the injection of a fluid, or mixture of fluids, into a cell culture chamber, according to particular embodiments;

[0066] DETAILED DESCRIPTION OF IMPLEMENTATION METHODS

[0067] The general principle of the present invention relates to the formation and in vitro culture of a three-dimensional tissue model (e.g., cells, spheroids, tumoroids, organoids, assembloids (i.e., combination of organoids, tumoroids and other biological constructs, or of organoids and tumoroids, or of organoids and other biological constructs), tissues, etc.), as well as a vascular system reproducing a physiological vascularization of said three-dimensional tissue model.

[0068] More specifically, the invention relates to the simultaneous in vitro formation and culture of a three-dimensional tissue model and a vascular system connected to a perfusion control system (hereinafter referred to as the "fluid control device") of the three-dimensional tissue model, for obtaining a vascularized three-dimensional tissue model. Irrigation of the three-dimensional tissue model by the perfusion control system via the vascular system generated according to the method of the invention ensures, in particular, the supply of nutrients and oxygen to the three-dimensional tissue model. A "vascularized three-dimensional tissue model" is understood to mean a three-dimensional tissue model comprising interwoven vascular and parenchymal tissues, the vascular tissues reproducing the physiological vascularization of the parenchymal tissue.

[0069] Hereafter, the term "cell" refers here to a mammalian cell (e.g., dog, cow, horse, sheep, rabbit, rat, mouse, human cell, etc.).

[0070] Figs. IA, IB, IC, and 1D schematically illustrate examples of implementation of a process for the formation and in vitro culture of a vascularized 3D tissue model according to embodiments of the invention.

[0071] Figs. 2A, 2B, 2C and 2D schematically illustrate examples of the arrangement of one or more cell culture chambers adapted to implement the process of forming and culturing, in vitro, a vascularized 3D tissue model according to embodiments of the invention.

[0072] A "cell culture chamber" (also referred to hereafter as a "culture chamber" or simply a "chamber") is understood to be a reservoir designed to hold liquid, solid, or gaseous products, and adapted to implement state-of-the-art cell culture techniques. Such a cell culture chamber may, where appropriate, be part of cell culture systems such as bioreactors or organ-on-chip microfluidic devices.

[0073] According to one embodiment, the cell culture chamber 100 has an internal volume between 1 mm 3 and 250 cm 3In one particular embodiment (see, for example, Fig. 2A, Fig. 2B), the chamber 100 may include one or more hermetic closure means 23 adapted to seal the chamber 100 hermetically and reversibly. These hermetic closure means 23 are located, for example, on an upper part of the chamber 100. They are removable, meaning that they can either allow access to the interior of the chamber 100 (e.g., for the introduction and / or manual removal of a fluid or mixture of fluids into the chamber 100, for access to biological samples, etc.) or seal it hermetically to isolate the interior of the chamber 100 from the external environment. Such hermetic closure means 23 are, for example, a lid or a cap. In another embodiment, the chamber 100 does not include any hermetic closure means 23 (see, for example, Fig. 2C).

[0074] In this case, chamber 100 is placed, for example, in an incubator to control the cell culture environment. This chamber 100 can then contain a predetermined volume (or quantity) of a fluid, or mixture of fluids, and / or a solid. By "fluid," we mean a liquid or gas with varying compositions, but also a liquid containing semi-solid materials (i.e., gel precursors, cells) or solids (i.e., metallic particles). By "mixture of fluids," we mean a mixture of several liquids with different compositions, a mixture of several different gases, or a mixture of liquid and gas (i.e., a liquid containing a predetermined quantity of dissolved gas, such as oxygen). "Solids" also include solidified or formed gels that trap or contain a significant quantity of fluids.It should be noted that chamber 100 can contain several predetermined volumes of different fluids, or mixtures of fluids, and / or solids. For example, chamber 100 can contain a first predetermined volume VI of a first fluid, or mixture of fluids (e.g., a liquid), and a second predetermined volume V2 of a second fluid, or mixture of fluids (e.g., a gas). In another example, chamber 100 can initially contain a first predetermined volume VI of a first fluid, or mixture of fluids, and a second predetermined volume V2 of a second fluid, or mixture of fluids (e.g., a gas). Then, subsequently, following, for example, a solidification process of a fluid to obtain a gel as described below, chamber 100 can contain a first predetermined volume VI of a solid (e.g., a gel), and a second predetermined volume V2 of a second fluid, or mixture of fluids (e.g., a gas or a liquid).

[0075] According to one embodiment (see, for example, Fig. 2A, Fig. 2C), the chamber 100 comprises one or more fluid inlets 10, 10', 20 and one or more fluid outlets 11, 11', 21. The fluid inlets 10, 10', 20 and the fluid outlets 11, 11', 21 are adapted to introduce, respectively, a fluid, or mixture of fluids, into / out of the chamber 100. The fluid inlets 10, 10', 20 and / or the fluid outlets 11, 11', 21 are, for example: an orifice (or opening) provided in a wall or the hermetic closure means 23 of said chamber 100, a tube, a microfluidic channel, or a nozzle passing through a wall or the hermetic closure means 23 of said chamber 100, or, in the case where the chamber is open at the top (i.e. no means of airtight closure 23), reaching the internal part of chamber 100 from its upper part and one of whose ends then opens into the interior of said chamber 100.According to a particular embodiment (see for example Fig. 2A, Fig. 2B, Fig. 2C), when the fluid inlets 10, 10', 20 and / or the fluid outlets 11, 11', 21 are tubes, microfluidic channels or nozzles, they can be positioned horizontally through a wall of the chamber 100 so that one of their ends opens into the interior of the chamber 100. Alternatively, when the fluid inlets 10, 10', 20 and / or the fluid outlets 11, 11', 21 are tubes, microfluidic channels or nozzles, they can be part of the hermetic sealing means 23 and are then positioned through the hermetic sealing means 23 of the chamber 100 so that one of their ends opens into the interior of the chamber 100.For example, chamber 100 includes one or more first fluid inlets 10, 10', one or more second fluid inlets 20, one or more first fluid outlets 11, 11', and one or more second fluid outlets 21. The first fluid inlet(s) 10, 10' and the first fluid outlet(s) 11, 11' are, for example, located in a lower part of a wall of chamber 100. The second fluid inlet 20 and the second fluid outlet 21 are, for example, located in an upper part of a wall of chamber 100, for example, near the hermetic sealing means 23 of chamber 100 or near the highest point of chamber 100. In a particular embodiment, the second fluid inlet 20 and the second fluid outlet 21 form part of the hermetic sealing means 23.According to this example, the first fluid inlet(s) 10, 10' are connected to one or more first control devices 12, 12' and the second fluid inlet(s) 20 are connected to one or more second fluid control devices 22. In an alternative, one or more or all of the fluid inlets 10, 10', 20 are connected to one or more conduits linking one or more fluid outlets from at least one other cell culture chamber (see Fig. 2D).

[0076] In one embodiment, the first fluid inlet(s) 10, 10' and the first fluid outlet(s) 11, 11' are immersed in the same first volume V1 of fluid or fluid mixture, while the second fluid inlet(s) 20 and the second fluid outlet(s) 21 are immersed in the same second volume V2 of fluid or fluid mixture, different from said first volume VI (see Fig. 2A). For example, the first fluid inlets 10, 10' and first fluid outlet(s) 11, 11' are immersed in a liquid or a solid, and the second fluid inlets 20 and second fluid outlet(s) 21 are immersed in a gas or gas mixture. "Immerse" means to place or insert a fluid outlet or a fluid inlet into a volume of fluid or fluid mixture.This immersion implies that the fluid inlet or outlet is in direct contact or partially submerged in the volume of fluid, or mixture of fluids.

[0077] It should be noted that an advantage of using a chamber 100 comprising a hermetic closure 23 and fluid inlets / outlets on the top of the chamber (i.e., for example through or integrated into the hermetic closure 23) is to maintain a predetermined pressure in the upper part of the chamber 100. In particular, it is possible to maintain a predetermined pressure on top of a liquid or solid (e.g., cell culture medium liquid or gel) contained in the chamber 100.

[0078] In one embodiment, there are several fluid inlets 10, 10' which are connected to a single fluid control device 12 (see, for example, Fig. 3B). In another embodiment, there are several fluid inlets 10, 10' which are connected to different fluid control devices 12, 12' (see, for example, Fig. 3C).

[0079] According to one embodiment, the fluid inlets and outlets have an inner diameter between 5 µm and 10 mm. Alternatively, the fluid inlets and outlets have cross-sections with an area between 16 µm 2 and 100 mm 2 It should be noted that each fluid inlet, and respectively fluid outlet, may have different dimensions relative to each other, preferably always within the values ​​described above.

[0080] According to one embodiment, the fluid inlet(s) 10, 10', 20 and / or the fluid outlet(s) 11, 11', 21 are each equipped with suitable closing means to: - prevent the circulation through the fluid inlet(s) 10, 10', 20 and / or the fluid outlet(s) 11, 11', 21 of a fluid, or mixture of fluids, when they are closed, - allow the circulation through the fluid inlet(s) 10, 10', 20 and / or the fluid outlet(s) 11, 11', 21 of a fluid, or mixture of fluids, when they are open.

[0081] Such means of closure include, for example, a valve or a backpressure device.

[0082] In one embodiment, the fluid inlet(s) 10, 10', 20 are connected, via at least one conduit (e.g., microfluidic channel), to one or more fluid outlets of one or more fluid control devices 12, 12', 22. The fluid control devices 12, 12', 22 are configured to adjust and / or regulate over time (i.e., control and modify if necessary) a predetermined flow rate of a fluid, or mixture of fluids, injected into the chamber 100 via the fluid inlet(s) 10, 10', 20 and / or a predetermined pressure of this fluid, or mixture of fluids, inside the chamber 100. In a particular embodiment, the fluid control devices 12, 12', 22 are further configured to control the opening and closing of the closing means equipping the fluid inlets 10, 10', 20 and / or fluid outlets 11, 11', 21.According to a particular embodiment, the fluid control devices 12, 12', 22 in combination with said closing means are further configured to guarantee, or maintain, a predetermined pressure, or pressure range, inside chamber 100, this pressure being measured in the upper part of chamber 100, and is preferably less than 100 bars at any given time.

[0083] Alternatively or additionally, the fluid control devices 12, 12', 22 are adapted to act as a means of closing the fluid inlet(s) 10, 10', 20, thereby preventing or allowing the flow through the fluid inlet(s) 10, 10', 20 of a fluid, or mixture of fluids.

[0084] According to one embodiment, the fluid, or mixture of fluids, injected by the control device(s) 12, 12', 22 into the chamber 100 includes gel precursors, or chemical or biochemical agents enabling the initiation of gel formation (e.g., crosslinking agents, polymerizing agents...).

[0085] In one embodiment, the fluid, or mixture of fluids, injected by the control device(s) 12, 12', 22 into chamber 100 is a cell culture and / or cell differentiation medium (i.e., a cell culture medium comprising compounds that enable cell differentiation). In another variant, the cell culture and / or cell differentiation medium further comprises cells of one or more cell types (e.g., endothelial cells, stem cells, circulating tumor cells, immune cells, etc.), and / or chemical or biochemical compounds (e.g., drugs, cell or gene therapy compounds, nanoparticles, exosomes, etc.).) intended to produce effects on tissues, i.e., assemblies of cells belonging to one cell type or assemblies of cells belonging to several different cell types and their extracellular matrix, and / or groups of tissues in the process of formation or already formed. In one embodiment, the fluid inlet(s) 10, 10', 20 are connected, via at least one conduit, to one or more fluid outlet(s) 11, 11', 21 of at least one other chamber (see, for example, Fig. 2D). This is the case, for example, when several chambers 100, 101, 102 are connected to each other in series, in parallel, or both (Fig. 2D).

[0086] In one embodiment, the culture chamber 100 allows for the cultivation of biological samples (e.g., cells, cellular tissues, etc.) through the injection and retention within the chamber 100 of various fluids, or mixtures of fluids, specifically adapted for cell culture, without contact with the external environment. Furthermore, the cell culture system according to the invention can control all cell culture parameters (e.g., mechanical signals, temperature, pH, nutrients, etc.) to guarantee a closed, or isolated, cell culture environment free from any external interference.The method for forming and culturing in vitro a three-dimensional vascularized cell model according to the invention is implemented in a system for forming and culturing in vitro a three-dimensional vascularized cell model (hereinafter referred to as the system) comprising at least one chamber, at least one fluid control device, and, where applicable, at least one temperature control device as described below according to one or a combination of the embodiments described.

[0087] By way of illustration and without limitation, it is hereafter considered that said at least one chamber 100 comprises two fluid inlets 10, 20 and two fluid outlets 11, 21 (Fig. 2A). The first fluid inlet 10 is connected to a first control device 12 and the second fluid inlet 20 is connected to a second fluid control device 22. However, it should be noted that a chamber 100 according to any one or a combination of the embodiments described above can be used for implementing the process according to the invention.

[0088] First, during a preliminary step, referred to as the "preparation of the culture chamber 100," a predetermined volume of a fluid, or mixture of fluids, referred to as the "first fluid 17," compatible with the culture and / or differentiation of various cell types, is introduced into the culture chamber 100. Subsequently, to illustrate the implementation of the process according to the invention, the predetermined volume of the first fluid 17 introduced into the chamber 100 is such that the first inlet 10 and outlet 11 of the fluid are immersed in said first fluid 17, while the second inlet 20 and outlet 21 of the fluid are not in contact with said first fluid 17 (Fig. 2A to Fig. 2D). The chamber 100 therefore comprises a first volume VI corresponding to a volume of the first fluid 17 and a second volume V2 free of first fluid 17 and which may contain another fluid, or mixture of fluids, 24.It should be noted, however, that the first predetermined volume VI of first fluid 17 introduced into chamber 100 can also correspond to the total volume of chamber 100 (Fig. 1A to Fig. 1D).

[0089] According to one embodiment, the introduction of this first fluid 17 is carried out automatically, that is to say by injection of this first fluid 17 by the first or second fluid control device 12 or 22 via the first fluid inlet 10 or the second fluid inlet 20. In this case, the chamber 100 is hermetically closed by the hermetic closure means 23 of the chamber 100 and the closure means of one of the fluid inlets 10 or 20 and of the second fluid outlet 21 (the closure means of the first fluid outlet 11 are then closed) are opened to allow the first fluid 17 to enter the chamber 100. The advantage of this automation is minimal operator interaction, which offers a high degree of repeatability in the creation and use of vascularized three-dimensional tissue models according to the invention.

[0090] Alternatively, the introduction of this first fluid 17 is carried out using a chamber 100 without a hermetic seal (Fig. 2C) or by removing the hermetic sealing means 23 from the chamber 100 to introduce the first fluid 17 manually or using a robotic mechanism (e.g., by pipetting). If applicable, once the first predetermined volume VI of the first fluid 17 is introduced into the chamber 100, the chamber can be hermetically sealed by the hermetic sealing means 23 (Fig. 2A and Fig. 2B). The first fluid inlet 10 and outlet 11, as well as the second fluid inlet 20 and outlet 21, are closed by the sealing means (i.e., sealing means closed) to prevent any leakage of the first fluid 17 during its introduction.

[0091] This first fluid 17 is intended to solidify into a gel 19, that is, a structure formed by a network of three-dimensional, semi-rigid, cross-linked polymers and a solvent, which does not flow in its steady state, after a transition through intermediate stages of semi-solidified material 18 (hereafter referred to as the "first fluid 17 during solidification 18"). It should be noted that a fluid, or mixture of fluids, can always diffuse through this gel 19, which, in the most relevant cases for our application, is then capable of absorbing liquids such as water, aqueous solutions, and biological fluids. To this end, the composition of the first fluid 17 includes gel precursors that enable its solidification through chemical cross-linking processes (e.g., ionic cross-linking, hydrogen bonding, etc.) or physical cross-linking processes (e.g., irradiation with visible / UV light, modification of temperature or pH, etc.).) known from the state of the art.

[0092] According to one embodiment, the second fluid control device 22 is configured to control the opening and closing of the second fluid inlet 20 and the second fluid outlet 21, for the injection of another fluid, or fluid mixture, different from the first fluid 17 and in direct contact with the upper surface of said first fluid 17 contained in the chamber 100 (i.e., the top of the first fluid 17). This fluid, or fluid mixture, thus injected by the second fluid control device 22 is hereafter referred to as the "third fluid 24" (Fig. 2C). In one example, this fluid, or fluid mixture, is a liquid composition comprising a cell culture and / or differentiation medium including predetermined factors (e.g., chemical or biochemical agents) enabling the crosslinking of the first fluid 17 and its solidification to obtain, from the gel precursors, a gel 19.It should be noted, however, that such a fluid, or mixture of fluids (i.e., third fluid 24), including, where appropriate, predetermined factors (e.g., chemical or biochemical agents) enabling the solidification of the first fluid 17, can also be introduced manually or using a robotic mechanism (e.g., by pipetting). In another example, alternatively or additionally (i.e., before and / or after the injection of the aforementioned liquid composition), the fluid, or mixture of fluids, is a gaseous composition comprising a predetermined concentration and / or percentage of one or more predetermined gases (e.g., CO2, N2, O2) or a liquid composition with a predetermined concentration of one or more dissolved gases or a predetermined pH.This gaseous or liquid composition can then be injected by the second fluid control device 22 until a predetermined pressure is reached inside the chamber 100, before and / or during and / or after the solidification process of the first fluid 17. This pressure can be maintained, for example by the second fluid control device 22, during the solidification process by controlling the injection (i.e., by controlling the flow rate and / or pressure) of the fluid, or fluid mixture, and by measuring the pressure, for example using sensors configured to measure such pressure. It is thus possible to apply a predetermined pressure of a predetermined gaseous or liquid composition to the surface of the first fluid 17, in particular, during its solidification, in order to control the rigidity and mechanical properties of the gel 19 obtained at the end of the solidification process.

[0093] According to a preferred embodiment, the first fluid 17 is solidified by a physical crosslinking process known from the prior art. Preferably, the physical crosslinking process used to solidify the first fluid 17 is based on modifying a predetermined factor, such as a temperature or temperature range, of the first fluid 17. Such a physical crosslinking process has the advantage of being easy to apply in a controlled manner to the first fluid 17 and, by implementing suitable parameters and using appropriate gels, of being compatible with biomolecules and living cells as known from the prior art. Furthermore, such a physical crosslinking process allows for a high degree of local adaptation of the physicochemical properties of the gel (e.g., stiffness) during its solidification (first fluid being solidified 18) and after its solidification (solidified gel 19) in response to local changes (e.g.pH and temperature, naturally generated by cells, can be part of the composition of the first fluid 17, in the same way as in a physiological or pathophysiological environment. This biomimicry is very important since cells are known to constantly remodel their local extracellular matrix (ECM) and, conversely, they can also respond to the mechanical and biochemical properties of their immediate environment.

[0094] Thus, in the preferred case, the gel precursors included in the composition of the first fluid 17 are, in particular, thermosensitive components or a combination thereof (e.g., hyaluronic acid, collagen, agar, agarose, gelatin, chitosan) enabling a time-dependent transition of the first fluid 17 from a liquid to a solid state at a predetermined temperature and at predetermined pH values ​​and concentrations of these thermosensitive components. Preferably, the gel precursors are chosen to allow solidification of the first fluid 17 under physiological or pathophysiological conditions known to be present in the cell types used, for example, when the temperature is modified to be within a range of 35°C to 38°C, preferably at 37°C.Furthermore, these gel precursors facilitate the inclusion, reproduction, migration, and subsequent survival of cells included in the structure of the gel being formed 18 (i.e., solidifying) and the gel 19 formed. In one particular embodiment, the gel 19 formed after solidification of the first fluid 17 is a hydrogel, that is, a material with a three-dimensional structure containing hydrophilic polymers capable of retaining a significant amount of water, aqueous solutions, and biological fluids. In a preferred embodiment, the hydrogel formed after solidification of the first fluid 17 is a hydrogel designed to closely mimic the native extracellular matrix, as well as extracellular molecules secreted by cells that provide structural and biochemical support to surrounding target tissue cells.Such hydrogels are, for example, formed using components of the extracellular matrix, thus creating so-called "naturally derived" hydrogels (e.g., Matrigel™, collagen, hyaluronic acid, methylcellulose, alginate, fibrin, or chitosan (a positively charged polysaccharide found in crustaceans)). In another example, such hydrogels can be made from synthetic polymers (e.g., Poly(ethylene glycol), Poly(vinyl alcohol), Poly(N-isopropylacrylamide), Poly(oligoethylene glycol methacrylate), Poly(2-hydroxyethyl methacrylate), etc.). In yet another example, it is also possible to mix naturally derived and synthetic hydrogels to form so-called "hybrid" hydrogels.

[0095] According to one embodiment (Fig. IA to Fig. 1D and Fig. 2A to Fig. 2D), the first fluid 17 further comprises cells or cell aggregates / tissues, and a cell culture medium suitable for the culture and / or differentiation of these cells or cell aggregates / tissues, and is initially maintained at a temperature, pressure and pH preventing the solidification of the first fluid 17 (i.e., gel formation) and cell death, typically at a pH between 6.5 and 8.5, a pressure below 200 kPa and a temperature between 5°C and 40°C, depending on the gel precursors used.It should be noted that the global and local temperatures of the first fluid 17, the first fluid in the process of solidification 18 or of the gel 19, as well as of the second fluid 13 described below, can be regulated directly by one or more or all of the fluid control devices 12, 12', 22 or by a temperature control device (not shown in the figures) configured to regulate and control the internal temperature of the chamber 100 and / or of the fluids, or mixture of fluids, injected into the chamber 100 by the fluid control device(s) 12, 12', 22. According to one embodiment (Fig. IA to Fig. ID, Fig. 2A to Fig.2D), these cells or cell aggregates / tissues are cells belonging to a cell type such as mature parenchyma stem cells from one or more target tissues to be mimicked or replicated, and / or cells belonging to a cell type such as mature stromal stem cells from the same target tissue to be mimicked or replicated, and / or from another tissue. In one example, the mature parenchyma stem cells of a target tissue to be mimicked or replicated include: hepatocytes, neurons, cardiomyocytes, and glial cells.

[0096] In one example, mature and / or stromal stem cells from a target tissue to be mimicked or replicated include: primary vascular cells, vessel fragments, stem cells, pluripotent or multipotent cells capable of vascular differentiation, vascular progenitor cells, endothelial cells, or immortalized cell lines. Preferably, the blood vessel-related cells include at least one of the following: primary endothelial cells, endothelial cell lines, primary pericytes, primary fibroblasts, pericyte lines, or fibroblast lines.Preferably, vascular stem cells are generated by the differentiation of mesodermal stem cells into vascular stem cells, the mesodermal stem cells having been obtained by stimulating mesodermal differentiation in pluripotent stem cells, using one of the known state-of-the-art methods.

[0097] According to a particular embodiment, the first fluid 17 comprises one or more cell groups, each comprising a set of cells belonging to one or more cell types from one or more tissues of interest to be imitated or replicated. In other words, the cells included in the first fluid 17 can be a mixture of any cell type of interest (e.g., stem cells, immune cells, etc.) intended to imitate or replicate a particular tissue. By way of illustration, the first fluid 17 is hereafter considered to comprise a first cell group 15, comprising a set of cells belonging to one or more cell types associated with the parenchyma of a target tissue to be imitated or replicated (e.g.mature cells and / or stem cells of the tissue parenchyma) and a second cell group 16 comprising a set of cells belonging to one or more cell types associated with the parenchyma of a target tissue to be imitated or replicated, tumor cells...). In a particular embodiment (Fig. IA, Fig. IC and Fig. 1D), the first fluid 17 further comprises a third cell group 14 comprising a set of cells belonging to one or more cell types associated with the stroma (e.g., mature cells and / or stem cells of the stroma).

[0098] These different cell types are co-cultured, allowed to differentiate and migrate before and / or during and / or after the solidification of the first fluid 17, which allows them to self-organize easily.

[0099] Next, in a step denoted E101, a second fluid 13 is injected by the first fluid control device 12 into the chamber 100 through the first fluid inlet 10. To do this, the fluid control device 12 commands the opening of the closing means of the first fluid inlet 10 and outlet 11 at a time t. The opening of the first fluid inlet 10 and outlet 11 simultaneously with the injection (i.e., introduction under pressure of a fluid) of the second fluid 13 by the fluid control device 12 causes a flow jet and establishes a laminar flow of the second fluid 13. A laminar flow is then generated between the first fluid inlet 10 and the first fluid outlet 11 through the first fluid 17 contained in the chamber 100. It should be noted that at this stage, the first fluid 17 is still in liquid form.It should be noted that the injection of the second fluid 13 is done either directly by the first control device 12, into the chamber 100, or by the introduction into a chamber 100 of the second fluid 13 from at least one other chamber (see Fig. 2D).

[0100] Laminar flow refers to the movement of a fluid in which it follows smooth, layered paths, each layer moving smoothly relative to adjacent layers with little or no mixing. Laminar flow is a flow regime characterized by high momentum diffusion and low convection.

[0101] According to one embodiment, the first fluid control device 12 is configured to maintain a predetermined flow rate of the second fluid 13 of less than 50ml / min.

[0102] Fig. 3A, Fig. 3B and Fig. 3C schematically illustrate examples of laminar flows generated by the injection of the second fluid 13 into the culture chamber 100 via one or more fluid inlets before and during the solidification process of the first fluid 17. Fig. 3A illustrates a particular example of laminar flow 30 flowing from a first fluid inlet 10 to a first fluid outlet 11 of the chamber 100.

[0103] Fig. 3B illustrates another particular example in which two laminar flows 30 and 31 are generated between a first fluid inlet 10 and a first fluid outlet 11 on the one hand, and between a second fluid inlet 10' and a second fluid outlet 11' on the other hand.

[0104] Fig. 3C illustrates another specific example in which two laminar flows 30 and 32 are generated between a first fluid inlet 10 and a second fluid inlet 10' and a single fluid outlet 11. In this particular example, the chamber 100 comprises two fluid inlets 10 and 10' and a single fluid outlet 11. The fluid inlets 10 and 10' are each arranged at a different height on the chamber 100 and separated from each other by a predetermined distance. The fluid outlet 11 is located opposite the chamber 100 and at a different height relative to the two fluid inlets 10 and 10'. In this example, the first fluid inlet 10 is connected to a first control device 12, and the second fluid inlet 10' is connected to a second fluid control device 12'. In one variant, the first and second fluid inlets 10, 10' are connected to a single fluid control device.In another variant, one or both of the fluid inlets 10, 10' are connected to one or more conduits linking one or more fluid outlets from at least one other cell culture chamber.

[0105] According to one embodiment, during this step E101 (i.e., before the initiation of solidification of the first fluid 17), the second fluid 13 injected into chamber 100 comprises, for example, a cell culture and / or cell differentiation medium (Fig. IA, Fig. IC, Fig. 1D). In one variant, the second fluid 13 further comprises cells from the third cell group 14 (i.e., mature and / or stromal-associated stem cells from a target tissue to be imitated or replicated) (Fig. IB, Fig. 1D). In another variant, it should be noted that, additionally or alternatively, the second fluid 13 may also comprise cells belonging to other cell types such as: stem cells, circulating tumor cells, immune cells, etc., and / or compounds intended to produce effects on the gels and / or tissues being formed (e.g., nanoparticles, exosomes, compounds modifying the structure of the gel, compounds altering the solidification process of the gel, predetermined solidification factors, ...) by penetration and / or diffusion in areas close to the interface 1000 between the first fluid 17 and the second fluid 13.

[0106] Then, during a step denoted El 02, the solidification of the first fluid 17 is initiated. The first fluid 17 is then in an intermediate state. The first fluid 17 is then said to be "the first fluid in the process of solidification 18".

[0107] According to one embodiment, to initiate the solidification of the first fluid 17, a predetermined factor such as temperature, or a range of temperatures, is modified to initiate the solidification of the first fluid 17 and obtain a gel 19. For example, the initial internal temperature of the chamber 100 is increased or decreased to reach a so-called "solidification temperature" between 35°C and 38°C, preferably 37°C. To this end, a temperature control device (not shown in the figures), configured in particular to adjust and / or regulate and control the internal temperature of the chamber 100, modifies the initial internal temperature of the chamber 100 in order to initiate the solidification of the first fluid 17. In a variant, alternatively or additionally, the predetermined factor may be the pH of the first fluid 17.The solidification of the first fluid 17 is then initiated in response to a change in the initial temperature, or initial temperature range, and / or the initial pH of the first fluid 17.

[0108] In another variant, the predetermined factor is a chemical or biochemical agent, or a mixture of chemical or biochemical agents, intended to enable the solidification of the first fluid 17 to obtain a gel 19. According to one example, the composition of the second fluid 13 can be modified at time t to initiate (at time t) the solidification of the first fluid 17 by incorporating said chemical or biochemical agent(s). According to another example, as described above, a third fluid comprising this chemical or biochemical agent(s) is injected into chamber 100 on top of the first fluid 17.

[0109] Thus, the initiation of the solidification of the first fluid 17 can be carried out by modifying one or more or a combination of the predetermined factors described above, i.e.: temperature or temperature range, pH, addition to the first fluid 17 of one or more chemical or biochemical agents intended to enable the solidification of the first fluid 17 to obtain a gel 19.

[0110] The laminar flow of the second fluid 13 is maintained at least during the solidification of the first fluid 17, i.e. for a predetermined duration allowing the formation process of the gel 19 to be completed in the chamber 100. In one example, this predetermined duration is less than 30 minutes.

[0111] An interface 1000 is created in the first fluid 17 before or during solidification 18. This interface 1000 between the first fluid 17 before or during solidification 18 and the laminar flow of the second fluid 13 is a zone in which there is a solidification rate gradient of the first fluid 17, as well as a strong diffusion of the components of the second fluid 13 (e.g., nutrients, dissolved gases (e.g., oxygen), temperature, etc.). By maintaining the laminar flow of the second fluid 13, it is possible to generate biophysical and / or biochemical stimuli at this interface 1000, such as a temperature gradient, shear stress, a concentration gradient of a gas or gas mixture, or a gradient of specific molecules (or chemical or biochemical compounds).The flow of the second fluid 13 during the solidification of the first fluid 17 prevents the formation of a gel 19 between the first fluid inlet 10 and the first fluid outlet 11 of chamber 100, while the first fluid 17 gradually solidifies around the laminar flow. A channel (or cavity, or fluid passage) passing through the first solidifying fluid 18 and extending from the first fluid inlet 10 to the first fluid outlet 11 is thus created.

[0112] In one particular embodiment, the temperature of the second fluid 13 is also adjusted and / or regulated, and controlled to reach a predetermined value between 5°C and 40°C. The adjustment and / or regulation, and control of the temperature of the second fluid 13 is carried out, for example, by the first fluid control device 12. Alternatively, or additionally, the temperature of the second fluid 17 can be adjusted and / or regulated and controlled by the temperature control device. It is thus possible to control the solidification rate of the first fluid 17 (i.e., accelerate or slow down solidification) by creating temperature gradients that are established between the channel created by the flow of the second fluid 13 and the first fluid being solidified 18.This temperature gradient then allows for the creation of a solidification rate gradient for the first fluid 17, which enables better control of gel formation and therefore the mechanical and structural properties of the gel 19. It is also possible to create temperature gradients near the vascularization, with implications for local cellular phenotypes. During a step labeled El 03, the solidification of the first fluid 17 is completed, and a gel 19 is obtained. The first fluid inlet 10 and the first fluid outlet 11 are then connected to each other through chamber 100 via the channel created by the flow of the second fluid 13 during the solidification of the first fluid 17.

[0113] Generally, the injection of the second fluid 13 by the first control device 12 is maintained after the complete formation of the gel 19. In particular, the composition and / or flow rate of the second fluid 13, circulating in the channel, are controlled and modified, if necessary.

[0114] Thus, according to one embodiment, during this step El 03, the composition of the second fluid 13 is modified (new composition of the second fluid 13) or a new second fluid replaces the second fluid 13 initially injected into chamber 100, for a predetermined period, to introduce and / or eliminate one or the other or a combination of the following elements:

[0115] - a cell culture and / or cell differentiation medium (Fig. IA) enabling the provision and maintenance of a pH, quantity of nutrients and dissolved gas composition (e.g., oxygen) such that it is possible to support or promote the maintenance, differentiation and / or reproduction of cells belonging to the first and second cell groups 15, 16, contained in the gel 19, and, where appropriate, to the third cell group 14, contained in the gel 19 or on the surface of the channel walls;

[0116] - cells of the third cell group 14 associated with the stroma of a target tissue to be imitated or replicated (Fig. IC);

[0117] - cells belonging to one or more other cell types (e.g., stem cells, circulating tumor cells, immune cells...);

[0118] - chemical or biochemical compounds (e.g., drugs, cell or gene therapy compounds, nanoparticles, exosomes, ...) intended to produce effects on tissues, i.e. assemblies of cells belonging to one cell type or assemblies of cells belonging to several different cell types and their extracellular matrix, and / or groups of tissues in the process of formation or already formed.

[0119] According to one embodiment, when the second fluid 13 initially comprises (i.e., before initiation of gel formation; see Fig. IB, Fig. 1D) or after modification of the second fluid 13 (i.e., after formation of gel 19; Fig. IC) cells of the third type Tl

[0120] Cells 14, then, after complete formation of the gel 19, the injection of the second fluid 13 is interrupted (e.g., by modifying the flow rate of the second fluid 13 by the first fluid control device 12, 12' such that the flow rate of the second fluid 13 is zero) for a predetermined period of less than 30 min, preferably 5 min. For this purpose, the closing means of the first fluid inlet 10 and the first fluid outlet 11 are closed. The channel then passing through the gel 19 is then filled with the second fluid 13 containing the cells of the third cell group 14. This allows these cells of the third cell group 14 to adhere to and fully or partially colonize the channel walls more effectively.Thus, controlling the flow rate of the second fluid 13 by the first fluid control device 12, 12', configured to inject it into the chamber 100, allows, for example, the cells of the third cell group 14 (e.g., endothelial cells) to attach more effectively to the wall(s) of the channel(s) in certain embodiments. In other embodiments, reducing the flow rate of the second fluid 13 by the first fluid control device 12, 12' for a specified period also increases the attachment of the third cell group 14 to the wall(s) of the channel(s).

[0121] It should be noted that, to facilitate colonization of gel 19 by cells of the third cell group 14, and thus the formation of microvessels 1002 in gel 19 and, ultimately, the vascularization of the 3D tissue model of interest, these cells are already included in the first fluid 17 in certain embodiments. Additionally, to facilitate colonization of the walls of the channel(s) created during the solidification of the first fluid 17, these walls are included in the second fluid 13 in certain other embodiments.By including the cells of the third cell group 14 in the first fluid 17 and in the second fluid 13 (before and / or during the formation of the gel 19), or only in the first fluid 17 or the second fluid 13, it is then possible to promote the colonization of the walls of the channel(s) and thus the obtaining of the coating 1001 and, in some cases, the formation of micro-vessels 1002.

[0122] After complete formation of the gel 19, and, where applicable, after allowing the cells of the third cell group 14 contained in the second fluid 13 to attach to the canal walls and colonize them totally or partially, the perfusion (or injection) of the second fluid 13 is maintained, changed, or, if necessary, resumed, for a predetermined period. This predetermined period is sufficient to allow the cells of the third cell group 14 contained in the gel 19 and / or in the second fluid 13 to migrate to the canal walls, to sporadically form vascular tissue, and / or to continue to be captured by the gel 19 at the canal walls. Furthermore, at the end of this predetermined period, the cells of the third cell group 14 included in the gel 19 and / or having attached to and / or being captured by the gel 19 at the canal walls (i.e.Cells of the third cell group 14 contained in the second fluid 13 multiply, generating internal vascular tissue within the gel 19 connected to the channel walls. In some cases, they change phenotype under the influence of the physical and biochemical microenvironment, and colonize the channel wall surface, either partially or completely, eventually reaching a sufficient level of confluence. Most importantly, the cells of the third cell group 14 attached to the channel walls, and those cells that self-organize to form vascular tubes attached to this channel, experience the biophysical stimulus of shear stress generated by the predetermined fluid flow. This is crucial for ensuring a good physiological phenotype of the resulting vascular tissue.It should be noted that the control of the flow of the second fluid 13 by the first fluid control device 12, 12' configured to inject it into the chamber 100, also allows the local pressures between this second fluid 13 circulating in the channel(s) and the gel 19 or the shear stress to be modified, which allows the culture, differentiation and phenotype of the cells to be controlled, by mimicking those physiological or pathophysiological during cell culture, for example.

[0123] The circulation of the second fluid 13 within the channel traversing gel 19 generates local pressure and shear stress (i.e., the tangential force of the circulating fluid on the surface of a material, causing its deformation) on the internal walls of the channel. Furthermore, the circulation of the second fluid 13 within the channel creates a diffusion gradient of nutrients and dissolved gases (e.g., oxygen) contained within the second fluid 13 between the channel walls and gel 19. Oxygen diffusion, local pressure, and local shear stress generate conditions that control the phenotype of the cell lining (e.g., the polarization of endothelial cells) and further promote the neogenesis of vasculature and vessels to and from the surrounding parenchymal cells formed and integrated within gel 19.It is thus possible to stimulate the differentiation and migration of cells from the third cell group 14 around the canal walls through the influence of biochemical and mechanical signals provided by the second fluid 13 circulating in the channel. The cells of the third cell group 14 then differentiate, generating a vascular lining 1001 around the channel. Simultaneously, vascular channels 1002 (or microvessels 1002) connected to the channel perfused by the first fluid control device 12 are generated de nuovo (i.e., angiogenesis), and penetrate the interior of the gel, typically after more than 24 hours.

[0124] The first and second cell groups 15 and 16 migrate, multiply, differentiate and organize themselves freely under the influence of biochemical and mechanical signals provided by the gel 19, such as a diffusion gradient of oxygen and biomolecules between the second fluid 13 and the gel 19, specific biomolecules or components contained in the gel 19, and pressure exerted by the second fluid 13 on the gel 19.

[0125] The in vitro production of such microvessels 1002 connected to a channel perfused by a first fluid control device 12, within, for example, tumoroid and assembloid models with tumoroids, is particularly advantageous for overcoming one of the current limitations of tumor models when applied, for example, to scientific research, therapy discovery, and therapy testing. This is also relevant, for example, when generating tissues mimicking or reproducing the central nervous system (CNS) because it allows modeling the blood-brain barrier where current models fail to study new therapies, as well as new in vivo therapy delivery systems targeting specific parts of the CNS.

[0126] During a step noted El 04, the cell culture conditions, including the composition of the second fluid 13 perfused into the channel, are regularly monitored and modified as necessary to continue the tissue culture and maturation process, depending on the cell types and tissues targeted (known state-of-the-art cell culture protocols).

[0127] The cells of the first, second and third cell groups 14, 15 and 16 then continue to organize, multiply and differentiate freely for a period generally between 24 hours and 60 days, preferably less than 7 days. A three-dimensional vascularized cell model 1010 corresponding to a combination of intertwined vascular tissue and parenchyma that can be perfused is then obtained at the end of the process according to the invention.

[0128] According to one embodiment, when several chambers 100 are interconnected, as described above, it is possible to obtain a network of several interwoven vascular and parenchymal tissues 1010, incorporated into the gels 19 of the different chambers 100 connected to each other by means of a network of conduits (e.g., tubes or microfluidic channels). This makes it possible to allow the exchange of elements (e.g., metabolites, exosomes, cells, etc.) produced by the different interwoven vascular and parenchymal tissues 1010. This more closely reproduces what occurs in natural physiological or pathophysiological tissues and is of paramount importance for developing new therapies based on a better understanding of inter-tissue communication.

[0129] According to one embodiment, before (step El 01) and / or during (step El 02) and / or after (steps El 03 and El 04) the complete formation of the gel 19, a fluid, or mixture of fluids, possibly exchangeable or renewable, referred to as the "third fluid 24," can be introduced into the upper part of the chamber 100. This fluid, or mixture of fluids, is then in direct contact with the upper surface of the first fluid 17 and / or the first fluid being solidified 18 and / or the formed gel 19. This fluid, or mixture of fluids, is, for example, introduced manually or robotically (e.g., by pipetting) as described previously.Alternatively, or additionally, the fluid, or mixture of fluids, can be introduced in an automated manner as described previously, i.e., via the injection of this fluid, or mixture of fluids, by the second fluid control device 22 via the second fluid inlet 20 in the upper part of the interior of the chamber 100. As described previously, this third fluid 24 can be a gaseous composition comprising a predetermined concentration and / or percentage of one or more predetermined gases (e.g., CO2, N2, O2) or a liquid composition with a predetermined concentration of one or more dissolved gases or a predetermined pH.The second control device 22 is then configured to inject this gaseous or liquid composition through the second fluid inlet 20 into the upper part of the chamber 100, until a predetermined pressure is reached inside the chamber 100, before and / or during and / or after the solidification process of the first fluid 17. It is thus possible to maintain the upper surface of the first fluid 17 and / or the first fluid being solidified 18 and / or the gel 19 formed in contact with this predetermined gaseous or liquid composition in order to facilitate the maintenance of a pressure, pH and dissolved oxygen mixture in the gel 19 to support or promote the maintenance, differentiation or reproduction of the cells and / or tissues included in the gel 19.According to a particular embodiment, when the third fluid 24 is a liquid composition, then the latter may further comprise, for example, a cell culture and / or cell differentiation medium for maintaining a pH, nutrient and / or biomolecule concentration, and dissolved oxygen concentration in the gel 19 that supports or promotes the maintenance, differentiation, or reproduction of the cells and / or tissues contained in the gel 19. Alternatively, or additionally, when the third fluid 24 is a liquid composition, the latter may comprise cells belonging to one or more other cell types (e.g., stem cells, circulating tumor cells, immune cells, etc.) and / or compounds intended to produce effects on developing or already formed tissues (e.g., drugs, cell or gene therapy compounds, nanoparticles, exosomes, etc.).) and / or cells belonging to the first cell group 15 and / or the second cell group 16.

[0130] According to a particular embodiment, after complete formation of the gel 19 and once the cells cultured according to the process of the invention (i.e., cells belonging to the first, second or third cell groups 14, 15, 16) reach a desired phenotypic expression (e.g., transcriptome, morphology...), the composition of the second fluid 13 is modified to introduce any cell culture medium comprising cells belonging to one or more other cell types (e.g., stem cells, circulating tumor cells, immune cells...) and / or chemical or biochemical compounds intended to produce effects on tissues in the process of formation or already formed (e.g., drugs, cell or gene therapy compounds, nanoparticles, exosomes...) to study, for example, the therapeutic effect of some of these components on these tissues.

[0131] According to a particular embodiment, after complete solidification of the first fluid 17 and formation of the gel 19, the composition of the liquid trapped in the gel 19 at the interface 1000 can be modified by adding chemical or biochemical compounds capable of altering the composition of the gel 19. Thus, the method and system according to the invention facilitate the transfer and introduction of cells of one or more cell types (e.g., endothelial cells, stem cells, circulating tumor cells, immune cells, etc.), and / or compounds intended to produce effects on developing or already formed tissues (e.g., drugs, cell or gene therapy compounds, nanoparticles, exosomes, etc.).) during the various stages described above, using the microvessels 1002 to introduce these agents into the vascularized 3D tissue model 1010, as occurs in natural physiology.

[0132] According to one embodiment (Fig. 2C), cells or cell / tissue aggregates 1011 such as: primary epithelial cells; epithelial fragments; pluripotent or multipotent stem cells capable of epithelial differentiation; epithelial progenitor cells, or immortalized epithelial cell lines, are cultured and / or differentiated using known state-of-the-art methods on the upper surface of the gel formed 19 manually or automatically (e.g., robotic pipetting, fluid control device 22, etc.). It is thus possible to generate more complex in vitro tissues with an epithelialized surface, such as skin, the choroid plexus in the brain, the respiratory tract, the intestines, or the kidneys, which may be necessary for testing or developing new therapies to treat diseases such as cancer, genetic and age-related degenerations of these organs.

[0133] In one embodiment, the fluid outlet(s) 11, 11', 21, and the hermetic sealing means 23 of the chamber 100 allow, in particular, access to biological samples (e.g., fluid, or mixtures of fluids, cells and / or tissues...) in contact with the gel 19 and / or circulating in the channel formed after solidification of the gel 19, for analytical purposes (e.g., genomic analysis, transcriptomic analysis, analysis of biological processes, screening of compounds...). For example, it is possible to access, for analytical purposes, the three-dimensional tissue model 1010 during its formation or already formed, during or after various stages of cell culture and / or modifications of the three-dimensional tissue model 1010 and / or application of compounds intended to produce effects on the tissues during their formation or already formed.

[0134] The method and system according to the present invention allow the study of a plurality of biological effects, particularly for the discovery and evaluation of therapeutic strategies. It is possible, among other things, to study angiogenesis, immune cell migration, test new drug candidates for immunotherapy or advanced therapeutic modalities (ATMs, drugs for human use based on gene therapy, cell therapy, and tissue engineering). The biological tissues obtained can be used to evaluate the efficacy, toxicity, dosage, and combined effects of therapies on human physiology and pathophysiology, as well as the toxicity of compounds, or to obtain valuable omics signatures at different experimental time points to better understand the physiological or pathophysiological mechanisms occurring in human tissues.In particular, the three-dimensional vascularized tissue model obtained using the method and system according to the invention is usable for studying a multitude of biological processes, the biological behavior of tissues and biological intercellular signaling, in processes such as the immune response (for example by introducing immune system cells into the composition of the second fluid 13 or the third fluid 24), neurodegenerative diseases or cancer.

[0135] The three-dimensional vascularized tissue model obtained using the method and system according to the invention can be used to identify, develop, and / or test candidate therapeutic agents or tissue regeneration strategies. It is particularly useful for therapies and strategies injected into the vascular system, but not exclusively. The case of the central nervous system (CNS) is especially relevant, as the development of effective therapies is severely hampered by the role of the blood-brain barrier (BBB) ​​and the strong capacity of this vascular system to prevent the passage of therapeutic agents or therapy carriers (e.g., nanoparticles) to reach diseased or degenerated CNS tissues, as well as by the role of the vascular system in the progression of unresolved pathologies such as glioblastoma or neurodegenerative diseases.In this sense, BBB formation requires strict conditions, including the presence of continuous flow in the developing vascular system, enabling constant interaction between induction and CNS signaling. The method and system according to the invention achieve this by producing a more realistic, three-dimensional vascularized tissue model and providing previously unavailable control over the stages of BBB genesis. This opens the door to reproducing a physiological CNS model incorporating the BBB. The method and system according to the invention also allow for spatial and temporal control of growth factors that define the lineage and specify morphogenesis, as those that induce vascular development can often be detrimental to other co-cultured tissue lineages, and vice versa.Indeed, the growth factors defining the lineage and specifying morphogenesis can be applied independently in the gel, in the vascular cavities defined by the gel (i.e., channel) and in the upper part of the gel.

Claims

DEMANDS 1. A method for the formation and in vitro culture of a vascularized three-dimensional tissue model, said method comprising: - introducing into at least one chamber (100) a first fluid (17) comprising gel precursors intended to form one or more three-dimensional networks under the influence of a predetermined factor or a combination of predetermined factors, said first fluid (17) further comprising cells belonging to at least a first cell group (15, 16) comprising cells belonging to one or more cell types of mature and / or stem cells associated with the parenchyma of one or more target tissues to be imitated or replicated, and / or at least a second cell group (14) comprising cells belonging to one or more cell types of mature and / or stem cells associated with the stroma,said method being characterized in that it further comprises: - initiating the culture and / or differentiation of said cells from said at least a first cell group and / or at least a second cell group, and, simultaneously, - injecting at least a second fluid (13) into said at least one chamber (100) containing said first fluid (17), by at least one first fluid control device (12, 12'), through at least one first fluid inlet (10, 10'), said at least one first fluid inlet (10, 10') being immersed in said first fluid (17), and said at least one second fluid (13) flowing from said at least one first fluid inlet (10, 10') to at least one first fluid outlet (11, 11') immersed in said first fluid (17), to create at least one flow jet and establish a laminar flow of said at least one second fluid (13) between said at least one first inlet of fluid (10,10') and said at least one first fluid outlet (11, 11'), - then, initiate a solidification of said first fluid (17) by modifying said predetermined factor or at least one of said predetermined factors of said combination to obtain a gel (19), while maintaining said laminar flow to prevent the formation of said gel (19) and create at least one channel between said at least one first fluid inlet (10, 10') and said at least one first fluid outlet (11, 11'), - maintain the culturing and / or differentiation of cells belonging to said at least one second cell group included in said first fluid and / or in said at least one second fluid until cells belonging to said at least one second group totally or partially colonize at least one wall of said at least one channel,- maintain the culture and / or differentiation of said cells belonging to at least one first cell group and at least one second cell group until said three-dimensional vascularized tissue model is obtained.

2. A method according to claim 1, characterized in that cells belonging to said at least one second cell group are included at least in said at least one second fluid (13) for a predetermined period before and / or during and / or after said solidification of said first fluid (17).

3. A method according to any one of claims 1 and 2, characterized in that it further comprises: adjusting over time a pressure exerted on the top of said first fluid (17) before and / or during and / or after said solidification of said first fluid (17).

4. A method according to any one of claims 1 to 3, characterized in that it further comprises: introducing at least one third fluid (24) into said at least one chamber (100), before and / or during and / or after said solidification of said first fluid (17), said third fluid (24) comprising one or a combination of the following elements: (i) cells belonging to said at least one first cell group; (ii) cells belonging to one or more other cell types; (iii) compounds intended to produce effects on some or all of the cells of said at least one first cell group and / or on some or all of the cells of said at least one second cell group and / or on some or all of the cells belonging to one or more other cell types; (iv) chemical or biochemical agents capable of initiating said solidification of said first fluid (17) and corresponding to said predetermined factor or at least to one of said predetermined factors of said combination; (v) a cell culture and / or cell differentiation medium suitable for the culture and / or differentiation of cells of said at least one first cell group and / or said at least one second cell group and / or said or said other cell types;(vi) a gas or a mixture of gases or a liquid comprising a predetermined quantity of one or more dissolved gases.; 5. A method according to any one of claims 1 to 4, characterized in that it further comprises: modifying a composition of said at least one second fluid (13) or replacing said at least one second fluid (13) with at least one other second fluid (13) for a predetermined period, after said solidification of said first fluid (17), to introduce and / or remove from said at least one second fluid (17) one or a combination of the following: (i) cells belonging to said at least one second cell group; (ii) cells belonging to one or more other cell types; (iii) compounds intended to produce effects on some or all of the cells of said at least one first cell group and / or on some or all of the cells of said at least one second cell group and / or on some or all of the cells belonging to one or more other cell types;(iv) chemical or biochemical agents capable of initiating said solidification of said first fluid (17) and corresponding to said predetermined factor or at least to one of said predetermined factors of said combination; (vi) liquids comprising predetermined quantities of gas or mixture of dissolved gases to produce at least one gradient of dissolved gas in the gel (19).

6. A method according to any one of claims 1 to 5, characterized in that said predetermined factor or at least one of said predetermined factors of said combination is: a temperature or a range of temperatures, the solidification of said first fluid (17) being then initiated by modifying the initial temperature of said first fluid (17) and / or of said at least a second fluid (13) to reach a predetermined temperature or temperature gradient at which the first fluid (17) gradually solidifies to obtain the gel (19).

7. A method according to any one of claims 1 to 6, characterized in that it further comprises: adjusting over time a flow rate of said at least one second fluid (13) generated by said at least one fluid control device (12, 12').

8. A method according to any one of claims 1 to 7, characterized in that it further comprises: adjusting over time a temperature of said at least a second fluid (13).

9. A system for the in vitro formation and culture of a vascularized three-dimensional tissue model, said system comprising: - a chamber (100) capable of containing a fluid, said chamber comprising at least a first fluid inlet (10, 10') and at least a first fluid outlet (11, 11'); and - at least one fluid control device (12, 12', 22) connected to said at least one first inlet; characterized in that said system is configured such that, when the chamber contains a first fluid (17) comprising gel precursors in liquid or in transition from a liquid to a solid state, said fluid control device is capable of injecting at least a second fluid (13) to form at least one flow jet and establish at least one laminar flow from said inlet to said outlet within said first fluid.

10. System according to claim 9, further comprising at least a second fluid inlet (20) connected to the chamber (100) and capable of introducing at least a third fluid (24) into said chamber.

11. System according to claim 9 or 10, wherein the fluid control device (12, 12', 22) is further configured to adjust over time a pressure exerted on said first fluid (17).

12. System according to any one of claims 9 to 11, wherein the fluid control device (12, 12', 22) is configured to selectively inject, into the chamber (100), one of a plurality of second fluids (13) having different compositions.

13. System according to any one of claims 9 to 12, further comprising at least one temperature control device configured to modify the temperature of said first fluid (17) in the chamber (100) in order to initiate its solidification.

14. System according to any one of claims 9 to 13, wherein the fluid control device (12, 12', 22) is configured to adjust the flow rate of at least one of said second or third fluids (13, 24).

15. System according to any one of claims 9 to 14, further comprising at least one temperature control device configured to adjust the temperature of at least one second fluid (13).

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