MULTI-COMPARTMENT ORGAN-ON-CHIP TO REPRODUCE AND FUNCTIONALIZE A TARGET ORGAN

Abstract

The present invention relates to a multi-compartment microfluidic device comprising a first cellular compartment (10) within which a target organ is reproduced, a second cellular compartment (20), located below the first cellular compartment (10) and separated from it by a porous membrane (30), and a third cellular compartment (40) connected to the first cellular compartment (10) by a means forming a biological interface (50). Figure 1

Description

Title of the invention: ORGAN-ON-CHIP MULTI-COMPARTMENT FOR REPRODUCTION AND TO FUNCTIONALIZE A TARGET ORGAN FIELD OF INVENTION

[0001] 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 according to the target organ whose function is to be reproduced in vitro for analysis and / or diagnosis, and customizable for each individual. STATE OF THE ART

[0002] Currently, organ-on-a-chip devices are known and represent a significant advance for research, particularly biomedical research and pharmacology. They are a promising emerging technology for personalized medicine, disease research, cosmetics, nutrition, and more. Indeed, these devices make it possible to test new molecules in a controlled environment without the need for animal testing or large-scale clinical trials, to study specific pathologies by reproducing the exact conditions under which they develop in the body, and to personalize treatments based on a given patient's cells, thus offering a more targeted and effective approach.

[0003] However, the devices currently available on the market also have drawbacks. A primary disadvantage is that they do not truly allow for individualized 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 reproduce 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 sense, some manufacturers have developed devices specific to a given organ, such as the Gut-on-chip model (developed by FLUIGENT to reproduce the functioning of the intestine) or the skin-on-chip model (developed by Microfluidics Innovation Center to reproduce the functioning of the intestine). 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 reproduce any target organ while 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 decisive advance in the field of organs-on-a-chip.

[0004] Consequently, current devices do not allow us to obtain results that take into account all the interactions of the target organ with its immediate environment, which does not allow us to reach satisfactory specific conclusions on a plurality of target organs. DESCRIPTION OF THE INVENTION

[0005] The inventors have developed, unexpectedly and surprisingly, an organ-on-a-chip type microfluidic device, making it possible to overcome all the problems mentioned above.

[0006] 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.

[0007] Thus, the present invention relates to a multi-compartment microfluidic device comprising:

[0008] - a first cellular compartment comprising epithelial cells of a target organ forming a layer capable of receiving and interacting with a biological sample of said target organ taken from an individual,

[0009] - a second cellular compartment, located below the first cellular compartment, including endothelial cells,

[0010] - a porous membrane intercalated between the first cellular compartment and the a second cellular compartment to facilitate physiological exchanges between the first cellular compartment and the second cellular compartment,

[0011] - a third cellular compartment comprising neurons distributed over a substrate comprising a plurality of electrodes configured to measure the functional activity of neurons, and

[0012] at least one means forming a biological interface to allow communication by neuronal connection between the first cellular compartment and the third cellular compartment.

[0013] The present invention therefore offers several advantages, including:

[0014] - a relevant reproduction of the target organ, the latter being then non-generic and personalized, within the first cellular compartment, of the target organ and its environment via the collection of a biological sample from said organ of the individual, without prior testing being necessary, said biological sample serving as a catalyst for the personalization of the device,

[0015] - increased functionalization of the target organ, on the one hand via the addition of the biological sample of an individual within the first cellular compartment and, on the other hand, via 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,

[0016] - an economical production since the reproduction of the target organ in the The first cellular compartment can be implemented in a generic and standardized manner.

[0017] - an expanded test panel since the device according to the invention makes it possible to measure The device measures 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 could, 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 allows for the measurement of the reciprocal effects between the biological environment of the first compartment (epithelial cells of the target organ and the biological sample) and the neurons of the third cellular compartment, whose role is to mimic the peripheral nervous system.

[0018] In the context of the 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 enabling the reproduction of biological interactions between various tissues or organs in a controlled environment. The compartments are interconnected, for example, by microfluidic channels, allowing the transfer of fluids, nutrients, or biological signals, thus simulating the natural physiological exchanges between these parts of the organism.

[0019] In the context of the invention, the term "cell compartment" refers to 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 (for example, porous membranes or microchannels), allowing controlled exchanges between them. Each cell compartment is designed to mimic a specific tissue or organ environment, allowing the culture of live cells under controlled conditions. Each cell compartment can accommodate cells of a particular type to reproduce the biological functions and interactions of various parts of the organism in vitro. It promotes biological interactions by replicating the natural physiological functions and exchanges of cells with other compartments, while ensuring physical separation.

[0020] In the context of the 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 exchanges 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 in the device allows us to study mucosal-specific cell interactions and to simulate important biological processes such as absorption, secretion, immune defense, and interactions with the microbiota.

[0021] In the context of the 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 an essential 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 (such as glandular cells), absorption (such as 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 various biological structures derived from the target organ, such as epithelial cells forming a multilayer, a spheroid, an organoid, a tissue, or an expiry. The epithelial cells used in the invention can be obtained from various sources, such as immortalized cell lines, allowing for continuous and stable culture in the laboratory, or from already differentiated primary cells. These cells can be implanted directly into an individual and then reimplanted to ensure the preservation of specific physiological characteristics. Alternatively, induced pluripotent stem cells (iPSCs), of human (hiPSC) or animal origin, can be 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, and secretion.

[0022] In the context of the invention, "multilayer" means a cellular structure composed of several superimposed layers of cells, enabling the reproduction of the architectural and functional complexity of biological tissues. In 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 in which epithelial cells divide and organize themselves into a multilayer.

[0023] In the context of this invention, "neurons" are defined as nerve cells specialized in the transmission of 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 metabolic center of the cell; the dendrites, branching extensions that receive signals from other cells; and the axon, a single extension often surrounded by a myelin sheath, which allows 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 scope 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 the 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 others. autonomic neurons, which control involuntary functions such as heart rate and digestion by relaying signals to internal organs.

[0024] In the context of the invention, "cell culture substrate" means a support, generally solid, that allows 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. Within the first cell compartment, the cell culture substrate must exhibit specific viscoelastic properties that mimic a supporting layer (such as a lamina propria layer in a mucous 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.

[0025] In the context of the invention, "natural or synthetic hydrogel" means 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 by chemical methods, using polymers such as alginate, polyethylene glycol (PEG), or photocrosslinkable polymers, allowing for precise modulation of their mechanical properties, porosity, and degradation time.

[0026] In the context of the invention, "naturally induced extracellular matrix from biological cells" means 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.

[0027] 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 adjust 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.

[0028] 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 the cells mimicking the target organ in the device. Humans are preferred for collecting these samples, but animals, particularly in the context of preclinical models or comparative studies, can also be used. These individuals serve as a biological source for adapting the in vitro cell models to the physiological and pathological specificities of each organism.

[0029] In the context of the invention, the term "means forming a biological interface to enable communication by neuronal connection between at least two cellular compartments" means a system comprising a contact junction between cell populations within said cellular compartments, facilitating communication via neuronal connections. This means includes neuronal cells (sensory neurons) of the third cellular compartment, enabling the transfer of biological information in the form of electrical or chemical signals between the third cellular compartment and the other cellular compartment with which it interacts.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, the porosity of which is advantageously between 10 nm and 40 pm and the pore density of which is advantageously between 10 and 1100 µm pores per cm2, advantageously between 1100 µm and 1100 µm pores per cm2; a porous capillary membrane, of polycarbonate, polyester, polyethylene terephthalate and / or polytetrafluoroethylene; a gel; a hydrogel and mixtures thereof.

[0030] In the context of the invention, "an intercalated porous membrane" means a microporous structure specially designed to allow the selective passage of essential substances, such as gases, nutrients, ions and fluids, between two distinct environments (between two cellular compartments for example). 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 species 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 the separate compartments.

[0031] 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.

[0032] In the context of the invention, "induced pluripotent stem cells" are defined as cells derived from adult somatic cells 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.

[0033] In the context of the invention, a "synthetic or natural mucus layer" is understood to be 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.

[0034] In the context of the invention, "stimulus" means 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.

[0035] Preferably, the present invention relates to a multi-compartment microfluidic device having the following technical characteristics, taken alone or in combination:

[0036] - 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;

[0037] - the epithelial cell layer is coated, at least partially, with a layer of synthetic or natural mucus;

[0038] - the first cell compartment comprises a cell culture substrate forming a surface on which rests the layer formed by the epithelial cells;

[0039] - 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;

[0040] - the cell culture substrate of the first cell compartment is further loaded with collagen, elastin, proteoglycans, glycosaminoglycans and / or in structural glycoproteins, which promotes cell growth, both epithelial cells and other cell types present;

[0041] - the concentration of collagen, elastin, proteoglycans, in glycosaminoglycans and / or glycoproteins of the structure of the cell culture substrate of the first cell compartment is between 1 and 10 mg / ml, preferably between 3 and 6 mg / ml;

[0042] - the cell culture substrate of the first cell compartment has a thickness between 5 nm and 200 |am;

[0043] - the cell culture substrate of the first cell compartment has a module of Young's modulus between 1kPa and 80kPa, preferably the Young's modulus of the cell culture substrate being between 3kPa and 15kPa;

[0044] - the endothelial cells are distributed over all the walls of the second cell compartment;

[0045] - the third cellular compartment and / or the biological interface forming medium rest(s) on a porous membrane, the latter being separate or being of 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 of said target organ of the individual;

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

[0049] - the individual is a human.

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

[0052] Advantageously, said 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. Brief description of the drawings

[0053] [Fig-1]: [Fig.1] is a schematic perspective representation of the device the invention according to a first embodiment;

[0054] [Fig.2]: [Fig.2] is an exploded schematic representation of the device according to the first embodiment of the invention shown in [Fig.1];

[0055] [Fig.3]: [Fig.3] is an exploded schematic representation of a first and second variant of the embodiment of the device according to the first embodiment of the invention shown in [Fig.1];

[0056] [Fig.4]: [Fig.4] is a schematic top-view representation of the device according to the first embodiment of the invention shown in [Fig. 1], of which an enlargement of the part illustrating the means forming a biological interface,

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

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

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

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

[0061] The present invention is illustrated in a non-limiting way by the following examples.

[0062] The device 1 according to the invention is intended to allow for a very high degree of personalization of the target organ of the patient to be 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.

[0063] To this end, the invention relates to a multi-compartment microfluidic device 1 comprising:

[0064] - a first cellular compartment 10 comprising epithelial cells 100 of a target organ forming a layer capable of receiving and interacting with a biological sample 101 of said target organ taken from an individual,

[0065] - a second cellular compartment 20, located below the first compartment cellular 10, comprising 200 endothelial cells,

[0066] - 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,

[0067] - a third cellular compartment 40 comprising 400 neurons distributed on a substrate 41 comprising a plurality of electrodes 44 configured to measure the functional activity of neurons, and

[0068] - at least one means 50 forming a biological interface to allow a communication via neuronal connection between the first cellular compartment 10 and the third cellular compartment 40.

[0069] 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.

[0070] The first cell 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 on a cell culture substrate 11. These cells 100 are cultured to form a monolayer of cells (Figures 6 to 8). It is therefore 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 said target organ, taken from an individual, directly onto the monolayer formed by the epithelial cells 100 ([Fig. 7]). According to the variant shown in [Fig.[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.

[0071] 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 other experimental conditions. Therefore, an inlet and an outlet 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 will be provided within the device.

[0072] According to another unrepresented variant 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.

[0073] 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.

[0074] The porous membrane 30, as shown in all the figures, corresponds to 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.

[0075] 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.

[0076] According to the variant of the invention shown in Figure 3B, the device 1 includes a porous membrane 30 not forming the electrode substrate 4L. This device 1 includes a separate membrane 31 forming said electrode substrate 4L. 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.

[0077] 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 means 50 forming the biological interface provided for this purpose, thereby allowing the epithelial cells 100 to be innervated and their response to given stimuli to be measured. The neurons 400 are distributed on a substrate 41 comprising a plurality of electrodes 44, which makes it possible to digitize the behavior of the epithelial cells 100, mimicking the target organ and its function, in response to said stimuli.

[0078] 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.

[0079] The device 1 according to the invention operates as follows.

[0080] 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 function and environment are to be reproduced are positioned within the first cellular compartment 10. 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 said 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.

[0081] In parallel, endothelial cells 200 are introduced and cultured within the second cellular compartment 20 with the aim of colonizing all of its walls, thus reproducing a blood vessel and allowing the device 1 according to the invention of ensuring a vascularization function of the target organ reproduced in the first cellular compartment 10.

[0082] In parallel, neurons 400 (for example, 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 the neuronal connections 401 enabling neuronal communication between the first cell compartment 10 and the third cell compartment 40. As already mentioned, these neurons 400 are distributed on a substrate 41 comprising a plurality of electrodes 44 allowing the functional activity of the neurons 400 to be measured.

[0083] Thus, when a particular stimulus is applied to one of the biological elements of the 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 by means of the electrodes 44 connected to a measuring device (not shown).

[0084] 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.

[0085] By way of alternative, 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 mechanical stimulus, such as a compressive force applied to the biological sample 101. It is thus possible to evaluate the reactions within the first cellular compartment 10, in particular the epithelial cells 100, in response to such a stimulus. Such an example can be reproduced for a physical stimulus, such as the application of a given temperature, a light source, or even of an electric current in order to evaluate its reactions within the first Cell compartment 10.

[0086] Reference list

[0087] 1: Multi-compartment microfluidic device

[0088] 2: Lid of the multi-compartment microfluidic device

[0089] 3: Base of the multi-compartment microfluidic device

[0090] 10 First cell compartment

[0091] 11 Cell culture substrate

[0092] 20 Second cell compartment

[0093] 21 Inlet of the second cell compartment

[0094] 22 Outlet of the second cell compartment

[0095] 30 Porous membrane

[0096] 31 Separate membrane incorporating the electrode substrate

[0097] 40 Third compartment

[0098] 41 Electrode substrate

[0099] 42 Inlet of the third cell compartment

[0100] 43 Outlet of the third cell compartment

[0101] 44 Electrode(s)

[0102] 45 additional electrode(s)

[0103] 50 means forming the biological interface between the first cellular compartment and the third cellular compartment

[0104] 51 fluidic microchannel

[0105] 100: cellsepithelial cells of the target organ

[0106] 101: biological sample of said target organ taken from an individual

[0107] 102: mucus layer

[0108] 200: endothelial cell(s)

[0109] 400: neuron(s)

[0110] 401: neural connection(s) [YES] 402: neuronal terminal(s)

Claims

Demands

1. A 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 epithelial cell layer (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 from 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 chosen from the list consisting of: - a chemical stimulus, - a biological stimulus, - a mechanical stimulus - a physical stimulus.

Images