Multi-compartment organ-on-a-chip for reproducing and functionalizing a target organ

Abstract

The present invention relates to a multi-compartment microfluidic device comprising a first cell compartment (10) within which a target organ is reproduced, a second cell compartment (20) located below the first cell compartment (10) and separated therefrom by a porous membrane (30), and a third cell compartment (40) connected to the first cell compartment (10) by a means forming a biological interface (50).

Description

[0001] TITLE: MULTICOMPARTMENTAL ORGAN-ON-CHIP TO REPRODUCE AND FUNCTIONALIZE A TARGET ORGAN

[0002] FIELD OF INVENTION

[0003] The present invention relates to the field of microfluidic devices that mimic the functions of organs (human or animal) at the microscopic scale. Such devices are also known as organ-on-a-chip. More specifically, the invention deals with a multi-compartment microfluidic device, adaptable to the target organ whose function is to be reproduced in vitro for analysis and / or diagnosis, and customizable for each individual.

[0004] STATE OF THE ART

[0005] Organ-on-a-chip devices are now well-established and represent a significant advancement for research, particularly in biomedical and pharmacological fields. They are a promising emerging technology for personalized medicine, disease research, cosmetics, nutrition, and more. These devices allow for the testing of new molecules in a controlled environment without the need for animal testing or large-scale clinical trials, the study of specific pathologies by replicating the exact conditions under which they develop in the body, and the personalization of treatments based on a given patient's cells, thus offering a more targeted and effective approach.

[0006] However, the devices currently available on the market also have drawbacks. A primary disadvantage is that they do not allow for true personalization for each patient because the cells used are not considered specific. Indeed, these commercially available cells may originate from a different genetic background than that of the patient whose organ is to be reproduced. Furthermore, these devices generally only replicate some of the functions of the target organ since not all interactions with the organ's environment are taken into account. For example, interactions with the microbiota specific to each organ (and each patient) or the vascular system are not considered in existing devices.In this respect, some manufacturers have developed organ-specific devices, such as the Gut-on-Chip model (developed by FLUIGENT to replicate the function of the intestine) or the Skin-on-Chip model (developed by Microfluidics Innovation Center to replicate the function of the skin). However, such devices are not adaptable to other organs with mucous membranes. Thus, there are currently no devices suitable for the nose, gums, vagina, or any other mucous membrane organ. Developing a device that could replicate any target organ, taking into account its entire environment, that could be easily adapted from one organ to another, and that could be specific to each patient would therefore represent a major advance in the field of organ-on-a-chip technology.

[0007] Therefore, current devices do not allow for results that take into account all the interactions of the target organ with its immediate environment, which does not allow for satisfactory specific conclusions to be drawn on a plurality of target organs.

[0008] DESCRIPTION OF THE INVENTION

[0009] The inventors have unexpectedly and surprisingly developed an organ-on-a-chip type microfluidic device, making it possible to overcome all the problems mentioned previously.

[0010] An objective of the present invention is to provide a microfluidic device enabling both optimal reproduction of the targeted organ and its interactions with its environment, optimized personalization of said organ for each individual and adaptability to different target organs.

[0011] Thus, the present invention relates to a multi-compartment microfluidic device comprising:

[0012] - a first cellular compartment comprising epithelial cells from a target organ forming a layer capable of receiving and interacting with a biological sample from said target organ taken from an individual,

[0013] - a second cellular compartment, located beneath the first cellular compartment, comprising endothelial cells,

[0014] - a porous membrane intercalated between the first cellular compartment and the second cellular compartment to facilitate physiological exchanges between the first cellular compartment and the second cellular compartment, - a third cellular compartment comprising neurons distributed on a substrate comprising a plurality of electrodes configured to measure the functional activity of neurons, and at least one means forming a biological interface to allow communication by neuronal connection between the first cellular compartment and the third cellular compartment.

[0015] The present invention therefore offers several advantages, including:

[0016] - a relevant reproduction of the target organ, which is then non-generic and personalized, within the first cellular compartment, of the target organ and its environment via a biological sample taken from said organ of the individual, without prior testing necessary, said biological sample taken serving as a catalyst for the personalization of the device,

[0017] - increased functionalization of the target organ, firstly through the addition of an individual's biological sample within the first cellular compartment and secondly through the reproduction of the effects of vascularization on said organ by the endothelial cells present in the second cellular compartment which interact with the first cellular compartment through the intercalated porous membrane,

[0018] - economical production since the reproduction of the target organ in the first cellular compartment can be implemented in a generic and standardized manner,

[0019] - an expanded range of tests, since the device according to the invention makes it possible to measure the reciprocal effects between the biological sample and the target organ to which it is applied, as well as the effects of other stimuli (e.g., nutrients, creams, medications, probiotics, etc.) internal or external to the first cellular compartment. An external stimulus to the first compartment may, for example, result from a deliberate modification within the second cellular compartment (e.g., stressing of endothelial cells), the effects of which are transmitted to the cells of the first cellular compartment via the intercalated porous membrane.Finally, the device according to the invention also makes it possible to measure the reciprocal effects between the biological environment of the first compartment (epithelial cells of the target organ and biological sample) and the neurons of the third cellular compartment whose role is to mimic the peripheral nervous system.

[0020] In the context of this invention, a "multi-compartment microfluidic device" is defined as a miniaturized device comprising several distinct compartments, each designed to house and culture cells of various cell types. Each compartment is designed to mimic a specific part of the organism, thereby reproducing biological interactions between different tissues or organs in a controlled environment. The compartments are interconnected, for example, by microfluidic channels, enabling the transfer of fluids, nutrients, or biological signals, thus simulating natural physiological exchanges between these parts of the organism.

[0021] In the context of this invention, a "cell compartment" is defined as a distinct section of a multi-compartment microfluidic device dedicated to the culture of specific cells. Each cell compartment is isolated from the others while being connected by biological interfaces (e.g., porous membranes or microchannels), allowing controlled exchanges between them. Each cell compartment is designed to mimic a particular tissue or organ environment, enabling the culture of live cells under controlled conditions. Each cell compartment can accommodate cells of a specific type in order to reproduce the biological functions and interactions of various parts of the organism under in vitro conditions. It promotes biological interactions by replicating the natural physiological functions and exchanges of cells with other compartments, while ensuring physical separation.

[0022] In the context of this invention, the term "target organ" refers to the specific organ of the organism that is to be reproduced in the first compartment of the multi-compartment microfluidic device. This target organ is mimicked by culturing appropriate cells that recreate the biological, anatomical, and physiological functions of the organ in question, thus enabling the study of its interactions with other body systems in an in vitro environment. Particular interest is given to mucous organs, which are covered by a layer of epithelial cells that secrete mucus and play a key role in exchange and protection against the external environment. These organs include, in particular, the intestines, colon, lungs, skin, stomach, nose, mouth, esophagus, vagina, urinary tract, genital tract, and rectum.Reproducing these organs within the device allows for the study of mucosal-specific cell interactions and the simulation of important biological processes such as absorption, secretion, immune defense, and interactions with the microbiota. For the purposes of this invention, "epithelial cells" refers to cells that form epithelial tissue, which constitutes the lining layer covering the internal and external surfaces of the body, including the skin, organs, blood vessels, and cavities. These cells play a crucial role as a protective barrier, separating the body from the external environment, limiting water loss, and protecting against infections or external aggressions.Epithelial cells are also known for their involvement in secretory functions (like glandular cells), absorption (like enterocytes in the intestine), and the transport of substances. They exist in various forms, such as squamous, cuboidal, or columnar, and can be organized into monolayers or multilayers (stratified layers), depending on their function and location in the body. They can originate from different biological structures derived from the target organ, such as epithelial cells forming a multilayer, a spheroid, an organoid, a tissue, or an expiant.The epithelial cells used in the invention can be derived from various sources, such as immortalized cell lines, allowing for continuous and stable culture in the laboratory; already differentiated primary cells taken directly from an individual and then reimplanted to ensure the preservation of specific physiological characteristics; or induced pluripotent stem cells (iPSCs), of human (hiPSC) or animal origin, which are reprogrammed to give rise to epithelial cells with the desired characteristics. Multilayered epithelial cells mean that, in culture, these cells form several successive layers to mimic the physiological barrier of the organ of origin. This multicellular structure reflects the natural morphology of epithelial cells in terms of protection, absorption, or secretion.

[0023] In the context of this invention, "multilayer" refers to a cellular structure composed of several superimposed layers of cells, enabling the reproduction of the architectural and functional complexity of biological tissues. Within the first cellular compartment, this multilayer can be formed on a cell culture substrate that must exhibit specific viscoelastic properties, allowing the recreation of an environment similar to that of the target organ, within which epithelial cells divide and organize themselves into a multilayer.

[0024] For the purposes of this invention, "neurons" are defined as nerve cells specialized in transmitting electrical and chemical signals within the nervous system. Generally, neurons are composed of three main parts: the soma, or cell body, which contains the nucleus and constitutes the cell's metabolic center; the dendrites, branching extensions that receive signals from other cells; and the axon, a single extension often surrounded by a myelin sheath, which enables the rapid transmission of electrical signals to other cells or muscles. These cells play a crucial role in communication and the coordination of biological functions by transmitting signals between different parts of the body and enabling the body's sensory and motor responses.Within the framework of the invention, particular interest is given to the types of neurons that can be integrated into one of the cellular compartments of the device according to the invention and that can develop in such a way as to innervate, via their respective nerve endings, one or more biological elements of another cellular compartment.Among these different types of neurons are sensory neurons, which transmit information from sensory receptors to the central nervous system for the perception of external stimuli such as pain, heat, or pressure; motor neurons, responsible for transmitting nerve signals to muscles to initiate voluntary and reflex movements; interneurons, which connect sensory neurons to motor neurons in the central nervous system, playing a relay and coordination role; and autonomic neurons, which control involuntary functions such as heart rate and digestion by relaying signals to internal organs.

[0025] For the purposes of this invention, "cell culture substrate" means a support, generally solid, that allows for the adhesion, growth, and proliferation of cultured cells. The substrate can be made of various materials, such as glass, treated plastic, or biomaterials, and is often coated with cell adhesion proteins (such as fibronectin, collagen, or laminin) to promote cell attachment. In the first cell compartment, the cell culture substrate must exhibit specific viscoelastic properties to mimic a supporting layer (such as the lamina propria in a mucosal organ), thus ensuring a physicochemical environment close to the physiological conditions of the target organ.This substrate plays a crucial role in regulating cell behavior, influencing processes such as morphology, differentiation, and intercellular signaling, and is suitable for research and cell culture applications, particularly in the form of multilayer epithelial cells present in the first cellular compartment.Thus, without limitation, the following structures can be cited among all the possible options: RGD Alginate (alginate functionalized with the RGD motif), IKVAV Alginate (alginate functionalized with the IKVAV motif), Mix (IKVAV Alginate + RGD Alginate), Collagen I, Modified Collagen (Collagen I + NGF + BDNF + Laminin 111), Modified Matrigel (Matrigel + PBS + NGF + BDNF), Modified MaxGel (humanized Matrigel + NGF + BDNF), TrueGel3D (PVA + Dextran + RGD motifs + NGF + BDNF), Acellular Lamina (ECM of decellularized dermal fibroblasts), Modified Matrigel + Laminin 111, Acellular Lamina + NGF + BDNF + Laminin 111, Acellular Lamina + Collagen I coating + NGF + BDNF + Laminin 111, MaxGel + NGF + BDNF + Laminin 111.

[0026] For the purposes of this invention, "natural or synthetic hydrogel" refers to a hydrophilic material capable of retaining a large amount of water within its structure while maintaining its shape, thanks to a three-dimensional network of polymers. Natural hydrogels are derived from biological sources, such as collagen, fibrin, hyaluronic acid, or agarose, and are often used for their biocompatible and biodegradable properties. In contrast, synthetic hydrogels are manufactured using chemical methods, employing polymers such as alginate, polyethylene glycol (PEG), or photocrosslinkable polymers, allowing for precise modulation of their mechanical properties, porosity, and degradation time.

[0027] In the context of this invention, "naturally induced extracellular matrix from biological cells" refers to a complex network of macromolecules and proteins produced by living cells that provides structural and biochemical support to tissues. This matrix is ​​composed of various components, such as collagen, glycoproteins, proteoglycans, and elastins, which interact dynamically with cells to regulate their behavior, proliferation, and differentiation. The naturally induced extracellular matrix plays a crucial role in tissue integrity and function by providing mechanical and chemical signals. By using biological cells to induce the formation of this matrix, a physiological environment that promotes cell interactions and biological exchange can be recreated.

[0028] For the purposes of this invention, a "biological sample of said target organ taken from an individual" means a sample of tissue, cells, or biomolecules from an individual's target organ, collected for the purpose of customizing and functionalizing the cells mimicking that organ in the device. This sample may include various types of biological samples, such as organ-specific cells, tissue fragments, biological fluids, or samples of associated microbiota. For example, a sample of gut microbiota or intestinal epithelial cells may be used to fine-tune and adapt the gut-mimicking cell cultures in the device, in order to more accurately reproduce the physiological functions and interactions of the target organ in a given individual.

[0029] For the purposes of this invention, "individual" means any living organism, human or animal, from which biological samples can be taken to personalize and functionalize cells mimicking the target organ in the device. Humans are the preferred source for collecting these samples, but animals, particularly in preclinical models or comparative studies, can also be used. These individuals serve as a biological source for adapting in vitro cell models to the specific physiological and pathological characteristics of each organism.

[0030] In the context of the invention, a "means forming a biological interface to enable communication via neuronal connections between at least two cellular compartments" is understood to mean a system comprising a contact junction between cell populations within said cellular compartments, facilitating communication via neuronal connections. This means includes neuronal cells (e.g., sensory, motor, autonomic, or glial neurons) from the third cellular compartment, enabling the transfer of biological information in the form of electrical and / or chemical signals between the third cellular compartment and the other cellular compartment with which it interacts. This means is designed to ensure strict fluidic isolation between the compartments, allowing for the application of differentiated treatments and the independent study of the effects on each cell population.Thus, according to specific embodiments, this means may include a microfluidic barrier or a microchannel structure sized to allow the selective passage of axons while maintaining the isolation of cell bodies, thereby ensuring directional and specific innervation. Advantageously, this means may include two plurality of microchannels separated from each other by a buffer compartment that contributes to the fluidic isolation of said means and in which axons can develop from a microchannel of the first plurality to a microchannel of the second plurality. Advantageously, the microchannels are configured to allow the passage of 1 to 50 axons each, while preventing the passage of cell bodies, even when the latter contract.This means may also include localized chemical gradients or growth factors to guide axonal growth, or be associated with functionalized substrates promoting neuronal adhesion or migration. This biological interface means advantageously comprises at least one of the elements selected from the group consisting of fluidic microchannels; PDMS (polydimethylsiloxane) microchannels; a porous membrane, advantageously with a porosity between 10 nm and 40 pm and a pore density between 10 and 1x10. A 9 pores per cm2, advantageously between lxO A 5 and lxlO A 9 pores per cm2; a porous capillary membrane, made of polycarbonate, polyester, polyethylene terephthalate and / or polytetrafluoroethylene; a gel; a hydrogel and mixtures thereof.

[0031] In the context of this invention, an "intercalated porous membrane" is defined as a microporous structure specifically designed to allow the selective passage of essential substances, such as gases, nutrients, ions, and fluids, between two distinct environments (for example, between two cellular compartments) while maintaining a physical barrier. This membrane can be composed of a uniform layer, a stack of layers, or an entanglement of fibers, often made from inert polymers (such as polycarbonate (PC), polyethylene terephthalate (PET), or polydimethylsiloxane (PDMS)), filamentous proteins (such as fibrin), or other materials. The pores or spacing of this membrane typically range from 0.1 to 10 µm, allowing the passage of molecules while preventing large solid particles larger than 1 µm from passing through.This membrane plays an important role in the reproduction or support of biological processes by mimicking the natural exchanges that occur across biological interfaces, thus promoting controlled transfer of substances while ensuring the integrity of separate compartments.

[0032] In the context of the invention, "functional activity of neurons" means the emission and propagation of a nerve message in the form of electrical signals and / or neurotransmitter secretions.

[0033] For the purposes of this invention, "induced pluripotent stem cells" are defined as cells derived from adult somatic cells that have been reprogrammed to regain a pluripotent state, similar to that of embryonic stem cells. These cells, commonly called iPSCs (induced pluripotent stem cells), have the capacity to differentiate into virtually any cell type in the body, including neuronal, cardiac, hepatic, or epithelial cells. Human induced pluripotent stem cells (hiPSCs) can be derived from adult human cells originating, for example, from organs of the digestive system, such as cells from the intestine, stomach, or colon.

[0034] In the context of this invention, a "synthetic or natural mucus layer" is defined as a viscous layer that typically covers the internal surfaces of certain organs and tissues, formed either from natural biological components or from artificial materials. The natural mucus layer is primarily composed of glycoproteins, such as mucins, as well as salts, water, and other macromolecules, which act to protect the underlying tissues from dehydration, pathogens, and irritants. This mucus also plays a role in the absorption and transport of substances across epithelial membranes, as well as in facilitating cell interactions. On the other hand, the synthetic mucus layer is created from artificial materials designed to mimic the functional and viscoelastic properties of the natural mucus layer.These materials may include synthetic polymers and hydrogels, which are formulated to provide lubrication, protection, and bioactivity similar to those of natural mucus.

[0035] For the purposes of this invention, a "stimulus" is defined as a specific agent or condition capable of eliciting a physiological response in a cellular, tissue, or organ environment, depending on its chemical, physical, mechanical, or biological nature. Stimuli thus include chemical stimuli such as hormones (e.g., insulin for regulating blood glucose), growth factors (e.g., epidermal growth factor EGF, which stimulates cell proliferation), and neurotransmitters (e.g., dopamine or serotonin for neuronal signaling). They also include mechanical stimuli, such as pressure (e.g., blood pressure exerted on vessel walls), elongation (e.g., the stretching of muscle cells), and substrate stiffness (the surface to which cells adhere, influencing their behavior).They also include physical stimuli, such as temperature (e.g., thermal variations that induce a heat stress response), light (e.g., light signals detected by cells), and electrostimulation (e.g., electrical impulses applied to nerve cells). Finally, they also incorporate biological stimuli, such as microorganisms (pathogenic or beneficial, e.g., bacteria or viruses triggering an immune response), and cytokines and chemokines produced in response to inflammation. These different categories of stimuli, acting alone or in combination, make it possible to reproduce a variety of physiological conditions and trigger specific responses in biological models.

[0036] Preferably, the present invention relates to a multi-compartment microfluidic device having the following technical characteristics, taken alone or in combination:

[0037] - said epithelial cells are derived from induced pluripotent stem cells, preferably said pluripotent stem cells originate from said individual, preferably they originate from the target organ of said individual from which said biological sample is taken;

[0038] - the layer of epithelial cells is covered, at least partially, with a layer of synthetic or natural mucus;

[0039] - the first cellular compartment comprises a cell culture substrate forming a surface on which rests the layer formed by the epithelial cells;

[0040] - the cell culture substrate of the first cell compartment comprises a natural hydrogel, a synthetic hydrogel or an extracellular matrix naturally induced from biological cells;

[0041] - the cell culture substrate of the first cell compartment is further loaded with collagen, elastin, proteoglycans, glycosaminoglycans and / or structural glycoproteins, which promotes cell growth, both epithelial cells and other cell types present;

[0042] - the concentration of collagen, elastin, proteoglycans, glycosaminoglycans and / or structural glycoproteins of the cell culture substrate of the first cell compartment is between 1 and 10 mg / ml, preferably between 3 and 6 mg / ml;

[0043] - the cell culture substrate of the first cell compartment has a thickness between 5 nm and 200 pm;

[0044] - the cell culture substrate of the first cell compartment has a Young's modulus between 1kPa and 80kPa, preferably the Young's modulus of the cell culture substrate being between 3kPa and 15kPa; - the endothelial cells are distributed over all the walls of the second cell compartment;

[0045] - the third cellular compartment and / or the means forming the biological interface rest(s) on a porous membrane, the latter being distinct from or being in one piece with the porous membrane intercalated between the first cellular compartment and the second cellular compartment;

[0046] - the first cellular compartment comprises said biological sample arranged on the layer formed by the epithelial cells;

[0047] - said biological sample is a microbiota sample from said target organ of the individual;

[0048] - the target organ is chosen from the list consisting of: the intestine, the colon, the lungs, the skin, the stomach, the nose, the mouth, the esophagus, the vagina, the urinary tract, the genital tract or the rectum;

[0049] - The individual is a human being.

[0050] The invention also relates to the use of a multi-compartment microfluidic device according to one of the variants of the invention to detect the effects of a stimulus on a target organ and / or on a biological sample.

[0051] Advantageously, the stimulus is chosen from the list consisting of: a chemical stimulus, a biological stimulus, a mechanical stimulus, and a physical stimulus.

[0052] Advantageously, the target organ is chosen from the following list: the intestine, colon, lungs, skin, stomach, nose, mouth, esophagus, vagina, urinary tract, genital tract, or rectum.

[0053] Figures:

[0054] [Fig. 1]: Figure 1 is a schematic perspective representation of the device of the invention according to a first embodiment;

[0055] [Fig. 2]: Figure 2 is an exploded schematic representation of the device according to the first embodiment of the invention shown in Figure 1;

[0056] [Fig. 3]: Figure 3 is an exploded schematic representation of a first and second variant embodiment of the device according to the first embodiment of the invention shown in Figure 1; [Fig. 4]: Figure 4 is a schematic top view of the device according to the first embodiment of the invention shown in Figure 1, including an enlargement of the part illustrating the means forming a biological interface.

[0057] [Fig. 5]: Figure 5 is a schematic cross-sectional representation, along the section plane AA shown in Figure 4, of the device according to the first embodiment of the invention shown in Figure 1;

[0058] [Fig. 6]: Figure 6 is a schematic cross-sectional representation, along the section plane AA shown in Figure 4, of the device according to the first embodiment of the invention shown in Figure 1, in which the biological elements have been reproduced schematically;

[0059] [Fig. 7]: Figure 7 is a schematic cross-sectional representation, along the section plane AA shown in Figure 4, of a third variant embodiment of the device according to the first embodiment of the invention;

[0060] [Fig. 8]: Figure 8 is a schematic cross-sectional representation, along the section plane AA shown in Figure 4, of a fourth variant embodiment of the device according to the first embodiment of the invention;

[0061] [Fig. 9]: Figure 9 shows two visualizations (Figures 9A and 9B) highlighting, by fluorescence, the cell cultures implemented in the device according to the invention. The second visualization (Figure 9B) highlights the innervation of the cell culture substrate of the first cell compartment;

[0062] [Fig. 10]: Figure 10 represents two visualizations (Figures 10A and 10B) highlighting the neurons, their neuronal connections (axons) and their neuronal terminals within the device according to the invention.

[0063] The present invention is illustrated in a non-limiting way by the following examples.

[0064] Device 1 according to the invention is designed to allow for a high degree of personalization of the target organ of the patient being tested, by functionalizing said organ thus reproduced. For the sake of clarity, the elements common to all representations of said device 1 according to the invention are not necessarily identified from one figure to another, although they are clearly present. To this end, the invention relates to a multi-compartment microfluidic device 1 comprising:

[0065] - a first cellular compartment 10 comprising epithelial cells 100 from a target organ forming a layer capable of receiving and interacting with a biological sample 101 from said target organ taken from an individual,

[0066] - a second cellular compartment 20, located below the first cellular compartment 10, comprising endothelial cells 200,

[0067] - a porous membrane 30 interposed between the first cellular compartment 10 and the second cellular compartment 20 to facilitate physiological exchanges between the first cellular compartment 10 and the second cellular compartment 20,

[0068] - a third cellular compartment 40 comprising neurons 400 distributed on a substrate 41 comprising a plurality of electrodes 44 configured to measure the functional activity of the neurons, and

[0069] - at least one means 50 forming a biological interface to allow communication by neuronal connection between the first cellular compartment 10 and the third cellular compartment 40.

[0070] As illustrated in particular in Figures 1 to 3B, the device 1 according to the invention has a casing shape delimiting a volume within which several distinct cellular compartments (10, 20, 40) coexist. Said volume defined by this device 1 is delimited by the lid 2 and the base 3.

[0071] The first cellular compartment 10, as shown in all the figures, corresponds to an open well in which epithelial cells 100 from the target organ, whose function is to be mimicked, are deposited onto a cell culture substrate 11. These cells 100 are cultured to form a monolayer of cells (Figures 6 to 8). It is then possible to continue culturing the cells 100 so that they form a more complex biological structure, such as a multilayer structure (not shown). Alternatively, and depending on the objectives, it is possible to place the biological sample 101 from the target organ, taken from an individual, directly onto the monolayer formed by the epithelial cells 100 (Figure 7).According to the variant shown in Figure 8, a layer of synthetic or natural mucus 102 can be intercalated between the layer of epithelial cells 100 and the biological sample 101 in order to protect the epithelial cells 100 against aggressions from elements of the biological sample 101, such as pathogens and irritants present in the microbiota or saliva.

[0072] According to an unshown embodiment of the invention, the first cell compartment may be in the form of a closed well, thus allowing for testing of other experimental conditions. Therefore, an inlet and an outlet will be provided within the device for introducing the various biological elements (epithelial cells, biological sample) and / or for renewing the culture medium necessary for the growth of the epithelial cells.

[0073] According to another variant not shown of the invention, the epithelial cells can be deposited directly on the bottom of the first cellular compartment 10. From then on, the porous membrane has a composition and thickness allowing the proliferation, within it, of the neuronal terminals 402 of the neurons 400, allowing the said epithelial cells 100 to be innervated and their response to given stimuli (nutrients, creams, drugs, probiotics, etc.) directly applied to the biological sample 101 or to the endothelial cells 200 mimicking vascularization.

[0074] The second cell compartment 20, as shown in all the figures, corresponds to a semi-closed well into which endothelial cells 200 are introduced via the inlet 22 and outlet 23 provided for this purpose. The endothelial cells 200 can thus be cultured in this second cell compartment 20 so as to colonize all the walls of this compartment, thereby mimicking a blood vessel and allowing the reproduction of vascularization. Such a compartment contributes to the functionality of the device 1 according to the invention. The second cell compartment 20 is located below the first cell compartment 10 and is separated from it by the porous membrane 30, thus allowing the epithelial cells 100 of the first compartment 10 to benefit from the effects of vascularization, generated by the endothelial cells 200, similar to effects present in vivo.

[0075] The porous membrane 30, as shown in all the figures, is a thin membrane interposed between the first cellular compartment 10 and the second cellular compartment 20 to facilitate physiological exchanges between these two compartments and their respective cells and other biological elements. According to the first embodiment of the invention (Figures 1, 2, and 4 to 8), the porous membrane 30 extends over the entire surface of the device 1 and also forms an electrode substrate 41 comprising a plurality of electrodes 44. This membrane 30 also forms the substrate on which the axons of the neurons 400 extend through the plurality of microchannels 51, thus forming the neuronal connections 401 of the means 50, which constitutes the biological interface enabling communication via neuronal connection between the first cellular compartment 10 and the third cellular compartment 40.

[0076] According to the variant of the invention shown in Figure 3A, the porous membrane 30 further comprises additional electrodes 45 present at the interface between the first cell compartment 10 and the second cell compartment 20. Such additional electrodes 45 allow, for example, the application of an electrical stimulus to the cells of the first cell compartment 10 and / or the second cell compartment 20 and the study of the cellular response to such a stimulus.

[0077] According to the variant of the invention shown in Figure 3B, the device 1 includes a porous membrane 30 not forming the electrode substrate 41. This device 1 includes a separate membrane 31 forming said electrode substrate 41. This independent membrane 31 further has an opening 32 located at the interface between the first cellular compartment 10 and the second cellular compartment 20, so as not to impact the physiological exchanges between these two compartments.

[0078] The third cellular compartment 40, as shown in all the figures, corresponds to a semi-closed well into which neurons 400 are introduced via the inlet 42 and outlet 43 provided for this purpose. The neurons 400 can thus be cultured in this third cellular compartment 40 and extend their respective neuronal terminals 402 into the first cellular compartment 10, via the microchannels 51 of the medium 50 forming the biological interface provided for this purpose, thereby allowing the innervation of the epithelial cells 100 and the measurement of their response to given stimuli. The neurons 400 are distributed on a substrate 41 comprising a plurality of electrodes 44, which allows the digitization of the behavior of the epithelial cells 100, mimicking the target organ and its function, in response to said stimuli.

[0079] The means 50, forming a biological interface to enable communication via neuronal connection 401 between the first cellular compartment 10 and the third cellular compartment 40 as shown in all the figures, corresponds to a plurality of fluidic microchannels 51 distributed side by side. According to an unshown embodiment of the invention, this means 50 may comprise, alternatively or cumulatively, other elements such as one or more PDMS microchannels, an additional porous membrane, a porous capillary membrane, a gel, a hydrogel, and mixtures thereof.

[0080] Device 1 according to the invention works as follows.

[0081] Within the first cellular compartment 10, epithelial cells 100 from the target organ (namely the intestine, colon, lungs, skin, stomach, nose, mouth, esophagus, vagina, urinary tract, genital tract, or rectum) whose function and environment are to be mimicked and whose environment is to be reproduced, are positioned. After these cells have colonized the entire surface of the first cellular compartment 10, in the form of a monolayer or a more complex biological structure such as a multilayer, a biological sample 101 from said target organ, taken from an individual, is positioned either directly onto the epithelial cells 100 or onto a layer of mucus 102 previously placed on these cells.The biological sample 101 in question is advantageously from the microbiota of the patient's target organ, thus enabling the biological system reproduced within the first cellular compartment 10 to be functionalized while personalizing it to the individual from whom said biological sample 101 was taken. Alternatively, depending on the intended purpose, said biological sample 101 taken may be a biological fluid, such as saliva.

[0082] In parallel, endothelial cells 200 are introduced and cultured within the second cellular compartment 20 in order to colonize all of its walls, thus reproducing a blood vessel and enabling the device 1 according to the invention to ensure a vascularization function of the target organ reproduced in the first cellular compartment 10.

[0083] Simultaneously, neurons 400 (e.g., sensory neurons) are introduced and cultured within the third cell compartment 40, with the aim of innervating the target organ reproduced in the first cell compartment 10, via their respective neuronal terminals 402. To achieve this, the axons of the neurons 400 grow through the fluidic microchannels 51, thus forming neuronal connections 401 that enable neuronal communication between the first cell compartment 10 and the third cell compartment 40 (Figures 9A to 10B). As previously mentioned, these neurons 400 are distributed on a substrate 41 comprising a plurality of electrodes 44 for measuring the functional activity of the neurons 400.

[0084] Thus, when a particular stimulus is applied to one of the biological elements of device 1, it is possible to evaluate the response of the epithelial cells 100 and, more generally, of all the biological elements present in the first cellular compartment 10 forming the target organ to be mimicked, to said stimulus via the functional activity of the neurons 400, which is measured via the electrodes 44 connected to a measuring device (not shown).

[0085] By way of non-limiting example of the use and operation of the device 1 according to the invention, it is possible to evaluate the impact of a chemical stimulus, such as a new molecule or a new composition, or of a biological stimulus, such as a pathogen (bacteria or virus which triggers an immune response) or a new nutrient, by applying it directly to the biological sample 101. Such a stimulus causes reactions within the first cellular compartment 10, in particular the epithelial cells 100. These reactions result in modifying the electrical activity of the neuronal terminals 402, which is recorded by the electrodes and then analyzed.Alternatively or cumulatively, the application of this chemical or biological stimulus can be indirect, by applying it within the second cellular compartment 20, which can then be transmitted to the cells of the first cellular compartment 10 via the intercalated porous membrane 30. This also allows for the evaluation of the influence of vascularization (e.g., the bioavailability of a new molecule) on such a stimulus.

[0086] As an alternative, non-limiting example of the use and operation of device 1 according to the invention, it is possible to evaluate the impact of a mechanical stimulus, such as a compressive force applied to the biological sample 101. It is thus possible to assess the reactions within the first cellular compartment 10, particularly the epithelial cells 100, in response to such a stimulus. This example can be replicated for a physical stimulus, such as the application of a given temperature, a light source, or an electric current, in order to evaluate the reactions within the first cellular compartment 10.

[0087] Finally, Figures 9 and 10 show fluorescence visualizations of cell populations within the device 1 according to the invention. For these visualizations, showing in particular the innervation capacity of the neuronal cells 400 of the third cellular compartment 40, via pluralities of microchannels 51 of the medium 50 forming the biological interface between the first cellular compartment 10 and the third cellular compartment 40, through which the neuronal connections 401 of the neurons 400 extend, the cells of the biological sample 101 and of the mucus layer 102 have been intentionally omitted.In Figure 9B, where the target organ epithelial cells 100 (in this case Caco-2 intestinal cells) have, moreover, been deliberately cultured so as not to cover the entire surface of the cell culture substrate 11 of the first cell compartment 10, it is possible to observe the innervation of said cell culture substrate 11, illustrated by the marked presence of neuronal terminals 402 within the first cell compartment 10.

[0088] List of references

[0089] 1: Multi-compartment microfluidic device

[0090] 2: Lid of the multi-compartment microfluidic device

[0091] 3: Base of the multi-compartment microfluidic device

[0092] 10: first cellular compartment

[0093] 11: Cell culture substrate

[0094] 20: second cellular compartment

[0095] 21: Entry of the second cellular compartment

[0096] 22: exit from the second cell compartment

[0097] 30: porous membrane

[0098] 31: Separate membrane incorporating the electrode substrate

[0099] 40: third compartment

[0100] 41: Electrode substrate

[0101] 42: entrance to the third cellular compartment

[0102] 43: exit from the third cellular compartment

[0103] 44: electrode(s)

[0104] 45: Additional electrode(s)

[0105] 50: medium forming the biological interface between the first cellular compartment and the third cellular compartment

[0106] 51: Fluidic microchannel

[0107] 100: epithelial cells of the target organ

[0108] 101: biological sample of said target organ taken from an individual

[0109] 102: mucus layer

[0110] 200: endothelial cell(s)

[0111] 400: neuron(s)

[0112] 401: Neural connection(s)

[0113] 402: neuronal terminal(s)

Claims

DEMANDS 1. Multi-compartment microfluidic device (1) comprising: a first cellular compartment (10) comprising epithelial cells (100) from a target organ forming a layer capable of receiving and interacting with a biological sample (101) from said target organ taken from an individual, a second cellular compartment (20), located below the first cellular compartment (10), comprising endothelial cells (200), a porous membrane (30) intercalated between the first cellular compartment (10) and the second cellular compartment (20) to facilitate physiological exchanges between the first cellular compartment (10) and the second cellular compartment (20), a third cellular compartment (40) comprising neurons (400) distributed on a substrate (41) comprising a plurality of electrodes (44) configured to measure the functional activity of the neurons (400),and at least one means (50) forming a biological interface to allow communication via neuronal connection (401) between the first cellular compartment (10) and the third cellular compartment (40).

2. Device (1) according to the preceding claim, wherein said epithelial cells (100) are derived from induced pluripotent stem cells, preferably said pluripotent stem cells are derived from said individual, preferably they are derived from the target organ of said individual from which said biological sample (101) is taken.

3. Device (1) according to any one of the preceding claims, wherein the layer of epithelial cells (100) is coated, at least partially, with a layer of synthetic or natural mucus (102).

4. Device (1) according to any one of the preceding claims, wherein the first cell compartment (10) comprises a cell culture substrate (11) forming a surface on which rests the layer formed by the epithelial cells (100).

5. Device (1) according to the preceding claim, wherein the cell culture substrate (11) of the first cell compartment (10) has a Young's modulus between 1kPa and 80kPa, preferably the Young's modulus of the cell culture substrate (11) being between 3kPa and 15kPa.

6. Device (1) according to any one of the preceding claims, wherein the endothelial cells (200) are distributed over all the walls of the second cellular compartment (20).

7. Device (1) according to any one of the preceding claims, wherein the first cellular compartment (10) comprises said biological sample (101) disposed on the layer formed by the epithelial cells (100).

8. Device (1) according to any one of the preceding claims, wherein said biological sample (101) is a microbiota sample of said target organ of the individual.

9. Use of a multi-compartment microfluidic device (1) according to any one of the preceding claims to detect the effects of a stimulus on a target organ and / or on a biological sample (101).

10. Use according to the preceding claim, wherein said stimulus is selected from the list consisting of: a chemical stimulus, a biological stimulus, a mechanical stimulus, a physical stimulus.

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