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

Abstract

The present invention relates to a multi-compartment microfluidic device comprising a first cell compartment (10) within which a target mucosa of a mucosal organ is reproduced and a second cell compartment (40) comprising neurons (400), the second cell compartment (40) being connected to the first cell compartment (10) by a means forming a biological interface (50) so as to enable communication via neuronal connection (401) between the first cell compartment (10) and the second cell compartment (40).

Description

[0001] TITLE: MULTICOMPARTMENTAL ORGAN-ON-CHIP TO REPRODUCE AND FUNCTIONALIZE MUCOUS CELLS

[0002] FIELD OF INVENTION

[0003] The present invention relates to the field of microfluidic devices that mimic the functions of mucous membranes (human or animal) in mucous organs (in particular, the mouth, nostrils, larynx, gastrointestinal tract, male or female genital tract, respiratory tract, eyes, and urethra) at the microscopic level. Such devices are also known as organ-on-a-chip. More specifically, the invention relates to a compact, adaptable, and customizable multi-compartment microfluidic device for each individual, enabling the reproduction, as close as possible to reality, of the in vitro functioning of mucous membranes in mucous organs, for the purpose of analysis and / or diagnosis of these specific organs. Such analytical and diagnostic capabilities are particularly useful in the pharmaceutical field.

[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. For all mucosal organs, these devices do not allow for a very accurate reproduction of all the physiological characteristics of the mucous membranes in question. For example, in the case of the mucous membranes of the respiratory, genital, or digestive systems, which are composed of several layers such as the mucosa, the submucosa, and sometimes other layers of connective and muscular tissue, no device on the market can effectively reproduce the relationships between these different layers or the physiological interactions with their immediate environment. More specifically, again in the case of a mucosa, the epithelial layer is often composed of specific epithelial cells, a layer of loose connective tissue called the lamina propria, and, in some organs, a thin muscular layer.In certain organs, such as the intestine or the nose, the mucosal surface may feature protruding structures, like intestinal villi or the cilia of the respiratory epithelium, which increase the functional surface area for absorption, filtration, or secretion. Beneath this mucosa, the submucosa, often rich in connective tissue, contains glands, blood vessels, lymphatic vessels, and nerves. The structural and functional specificities of these layers and their complex interrelationships are not fully accounted for in existing devices, as they are so difficult to reproduce.This is especially true for structures such as the lamina propria, whose composition (largely made up of an assembly of proteins such as collagen and elastin, as well as cells like fibroblasts), variable thickness (from a few micrometers to several millimeters depending on the organ), and viscoelasticity are complex parameters. Structurally and functionally reproducing such a layer, given its specific characteristics and its various interactions with other layers of the mucosa and the surrounding environment, represents an obstacle that no current device has yet been able to overcome.

[0007] Finally, currently available devices do not offer a compact model integrating both a culture of epithelial cells from one of the mucous membranes of mucous organs and other compartments to reproduce the effects of vascularization and / or innervation. For example, epithelial cells reproducing a mucous membrane, such as that of the intestine, cannot differentiate into physiological tissue without shear flow, while neuronal cells cannot tolerate this same shear flow. Furthermore, epithelial cells require extensive physiological exchange to develop, whereas neurons differentiate poorly in an environment too open to physiological exchange.Finally, neurons cannot properly develop their extensions (axons and dendrites) if epithelial cells are in culture above them, hence the need for compartmentalization involving strong architectural constraints to overcome in order to obtain a compact and functional device.

[0008] Developing a device capable of reproducing the complexity of any mucous membrane in a mucous organ, while taking into account its entire interaction environment, and which is adaptable to different organs, customizable for each patient, all-in-one, and extremely compact, would represent a major advance in the field of organs-on-a-chip. For example, it would become possible to evaluate the impact of a given molecule (in applications as varied as nutrition, cosmetics, pharmaceuticals, medicine, biology, diagnostics, or personalized research (B2C)), taking into account all the functions of the mucous membrane in question. Such an advance would also drastically reduce the need for preliminary animal testing, thus addressing an ethical constraint that has been difficult to overcome until now.

[0009] Therefore, current devices do not allow for results integrating all interactions of the target organ with its immediate environment, which prevents drawing satisfactory specific conclusions for a plurality of target mucous membranes and associated mucous organs.

[0010] DESCRIPTION OF THE INVENTION

[0011] 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.

[0012] An objective of the present invention is to provide a compact organ-on-a-chip type microfluidic device that very faithfully reproduces the targeted mucosa, both in its structural complexity and in its interactions with its environment, thus enabling the evaluation and testing of said targeted mucosa and the associated mucosal organ in the many applications mentioned.

[0013] Thus, the present invention relates to a multi-compartment microfluidic device comprising:

[0014] - a first cellular compartment comprising a biological structure derived from the mucosa of a mucous organ, including epithelial cells of said mucosa, a cell culture substrate forming a surface on which said biological structure is disposed and configured to promote the growth or quiescence of the epithelial cells (100) of the biological structure and to permit, through itself, physiological exchanges with said epithelial cells, - a second cellular compartment comprising neurons distributed on a cell culture substrate comprising a plurality of electrodes configured to measure the functional activity of the neurons, and

[0015] - at least one means forming a biological interface to allow communication via neuronal connection between the first cellular compartment and the second cellular compartment.

[0016] The present invention therefore offers several advantages, including:

[0017] - a highly relevant reproduction, since the targeted mucosa is reproduced within the first cellular compartment, notably mimicking the structure and interactional complexity of the lamina propria,

[0018] - increased functionalization of the target mucosa, firstly via the possibility of adding a biological sample from an individual within the first cellular compartment and secondly via the possibility of reproducing the effects of innervation and / or vascularization on said mucosa by, respectively, the neurons of the second cellular compartment and the endothelial cells present in the third cellular compartment,

[0019] - a generic, economical, and standardized device since it allows the culture of cells (particularly pluripotent stem cells) from commercially available cell lines, while also enabling the personalization of the reproduced organ through the possibility of adding a biological sample from an individual. Alternatively, and for even greater personalization, it is also possible to culture pluripotent stem cells from the same individual from whom the biological sample is taken.

[0020] - an expanded range of tests, since the device according to the invention allows for the measurement of a multitude of effects, for example, reciprocal effects between a biological sample and the target mucosa to which it is applied, and the effects of other stimuli (e.g., the application of nutrients, creams, medications, probiotics, etc.), whether 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 third 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.Other examples of external stimuli whose effects can be transmitted to the targeted mucosa (or a specific part thereof) include mechanical stimuli (such as a puncture), optical stimuli (application of light with a specific wavelength), and electrical or bioelectrical stimuli. 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 mucosa and the biological sample) and the neurons of the second cellular compartment, whose role is to mimic the peripheral nervous system.

[0021] 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.

[0022] 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.

[0023] In the context of this invention, the term "target mucosa" or "target mucosa of a mucous organ" refers to the specific mucosa of an organ that is to be reproduced in the first compartment of the multi-compartment microfluidic device. This target mucosa is mimicked by culturing appropriate cells that recreate the biological, anatomical, and physiological functions of the mucosa in question, thus enabling the study of its interactions with other body systems in an in vitro environment. The invention is of particular interest to the mucosa of the mouth, respiratory tract, genital tract, and organs of the digestive system, such as the intestinal, gastric, esophageal, and colonic mucosa. These mucosa are covered by a layer of epithelial cells that secrete mucus and play a key role in exchange and protection against the external environment.Reproducing these mucous membranes in the device allows us to study mucous-specific cell interactions and to simulate important biological processes such as absorption, secretion, immune defense, or interactions with the microbiota.

[0024] For the purposes of this invention, a "mucous organ" means any organ lined by a layer of mucous tissue that protects it and participates in various exchanges with the environment. Mucous organs include the mouth, nasal passages, respiratory tract (including the larynx and trachea), the entire digestive system (including the esophagus, stomach, small intestine and colon), as well as the organs of the genital tract (for example, the vagina and uterus).

[0025] For the purposes of this invention, a "biological structure derived from the mucosa of a mucous organ" means an entity formed from cells or tissues originating from the mucosa of a mucous organ, such as the mouth, nasal passages, respiratory tract, genital tract, or an organ of the digestive system like the esophagus, stomach, small intestine, or colon. These biological structures may include, but are not limited to, monolayer epithelial cells, multilayer epithelial cells, a spheroid, an organoid, a tissue, or an expiant. These entities are used to model the biological functions and interactions specific to mucous organs in an experimental or therapeutic context.As a non-limiting example, these structures (including monolayer epithelial cells, multilayer epithelial cells, spheroids or organoids) can be generated, at least partially, from induced pluripotent stem cells (iPSCs), derived and reprogrammed to mimic the specific characteristics of epithelial cells or other cell types specific to the mucous membranes of the target organs.

[0026] In the context 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 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 involved in secretory functions (such as glandular cells), absorption (such as enterocytes in the intestine), and the transport of substances. In the context of this invention, particular interest lies in the epithelial cells of the mucous membranes of mucous organs, which form the epithelial tissue constituting the protective lining layer of the mucous membranes of various organs.These cells line the internal surfaces of structures such as the mouth, nasal passages, respiratory tract, genital tract, and the digestive system, including the esophagus, stomach, small intestine, and colon. They play a vital role as a barrier against the external environment, participate in regulating water loss, and protect against infections. In addition to their protective functions, mucosal epithelial cells contribute to mucus secretion, nutrient absorption, and the transport of various substances. These cells can have various shapes, such as squamous, cuboidal, or columnar, and can be organized into monolayers or multilayers (stratified layers) depending on their location and specific function.They can originate from various biological structures derived from the target mucosa, 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. Advantageously, in the context of the invention, these epithelial cells form a multilayer in culture, making it possible to reproduce the physiological barrier of mucous membranes.This multicellular organization replicates the morphology and natural functions of mucosal epithelial cells in terms of protection, mucus secretion, and absorption. These cells can be, for example, squamous, salivary, or olfactory epithelial cells, depending on the type of mucosa targeted.

[0027] 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 skin model, within which epithelial cells divide and organize themselves into a multilayer.

[0028] In the context of this invention, a "spheroid" is defined as a three-dimensional structure composed of an aggregate of cells, primarily epithelial, that spontaneously organize themselves into a spherical shape. This structure makes it possible to partially recreate natural cell interactions as well as cell differentiation and behavior in vivo, particularly with regard to growth, migration, and response to external stimuli such as drugs, nutrients, or microbial agents (pathogenic or beneficial).

[0029] For the purposes of this invention, an "organoid" is defined as a three-dimensional structure derived from stem cells or primary cells that functionally and structurally reproduces certain characteristics of the mucous membrane of a mucous organ. An organoid reflects several aspects of the architecture and specific functions of the original mucous membrane, such as enzyme secretion, nutrient absorption, or immune response, and as such constitutes a biomimetic model.

[0030] In the context of this invention, "tissue" means an organized assembly of several cell types from the mucous membrane of a mucous organ, cultured in the laboratory from biological samples. This tissue reproduces the interactions between epithelial cells and supporting or immune cells, and is used in particular to study the physiology and pathologies of the mucous membranes of mucous organs in a controlled environment.

[0031] For the purposes of this invention, "expiant" means a tissue fragment taken directly from an individual, whether human or animal. This sample retains the native architecture and function of the tissue and cells, making it possible to model biological and pathological processes under experimental conditions closely resembling the in vivo physiological state. An expiant can, in particular, be used to observe tissue interactions, responses to treatments, and physiological exchanges within a tissue derived from the mucosa that one seeks to reproduce. For the purposes of this invention, "neurons" means nerve cells specialized in the transmission of electrical and chemical signals within the nervous system.In general, neurons are composed of three main parts: the soma, or cell body, which contains the nucleus and is 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 allows for 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 ending, 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.

[0032] 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 that mimic the lamina propria layer, thus ensuring a physicochemical environment close to the physiological conditions of the target mucosa.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 from 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.

[0033] 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.

[0034] 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.

[0035] For the purposes of this invention, "promoting the growth or quiescence of epithelial cells" refers to all the conditions and factors that influence the development, proliferation, and maintenance of epithelial cells in an active or resting state. Epithelial cell growth refers to their ability to divide, migrate, and organize themselves to form functional layers, while quiescence refers to a resting state in which cells are metabolically active but not dividing.

[0036] For the purposes of this invention, "promoting physiological exchanges with epithelial cells" refers to all the mechanisms and conditions that allow epithelial cells to interact effectively with their environment and exchange substances essential for their function. These physiological exchanges may include nutrient diffusion, molecule absorption, waste elimination, and cell communication via chemical signals and signaling molecules. These interactions are crucial for maintaining homeostasis and proper tissue function.

[0037] For the purposes of this invention, a "biological sample of a mucous organ, relating to said mucous membrane, taken from an individual" means a sample of tissue, cells, or biomolecules from an individual's mucous organ, collected for the purpose of customizing and functionalizing the mucous membrane-mimicking cells in the device. This sample may include various types of biological samples, such as mucous-specific cells, tissue fragments, biological fluids, or samples of associated microbiota. For example, a sample of oral microbiota or nasal epithelial cells may be used to fine-tune and adapt the mucous membrane-mimicking cell cultures in the device, in order to more accurately reproduce the physiological functions and interactions of the mucous organ in a given individual.

[0038] 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 mucosa in the device. Humans are preferred for collecting these samples, but animals, particularly in preclinical models or comparative studies, can also be used. These individuals serve as a biological source to adapt the in vitro cell models to the specific physiological and pathological characteristics of each organism.

[0039] 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 second cellular compartment, enabling the transfer of biological information in the form of electrical and / or chemical signals between the second 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 directionality and specificity of 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.

[0040] 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 microns, allowing the passage of molecules while preventing the passage of large solid particles larger than 1 micron.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.

[0041] 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.

[0042] 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 the mouth, nasal passages, respiratory tract, organs of the digestive system, such as cells from the intestine, stomach, or colon, or organs of the reproductive tract, such as cells from the vagina or uterus.

[0043] 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.

[0044] 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.

[0045] Preferably, the present invention relates to a multi-compartment microfluidic device having the following technical characteristics, taken alone or in combination:

[0046] - the biological structure derived from a mucous membrane of a mucous membrane organ is chosen from the list consisting of: epithelial cells forming a monolayer, epithelial cells forming a multilayer, a spheroid, an organoid or a tissue or an expiant;

[0047] - the epithelial cells of the biological structure derived from a mucous membrane of a mucous organ cover the entire surface of the cell culture substrate; - the epithelial cells are squamous, salivary or olfactory epithelial cells and / or are derived from induced pluripotent stem cells, preferably human induced pluripotent stem cells;

[0048] - epithelial cells are chosen from: epithelial cells from oral mucosa, epithelial cells from nasal mucosa, epithelial cells from respiratory mucosa, epithelial cells from the mucosa of an organ of the digestive system and epithelial cells from the mucosa of an organ of the genital tract;

[0049] - epithelial cells are chosen from: laryngeal mucosal epithelial cells, tracheal mucosal epithelial cells, intestinal mucosal epithelial cells, stomach mucosal epithelial cells, esophageal mucosal epithelial cells or colon mucosal epithelial cells, vaginal mucosal epithelial cells and uterine mucosal epithelial cells;

[0050] - the cell culture substrate of the first cell compartment comprises a plurality of protrusions protruding from the surface of said cell culture substrate;

[0051] - the surface of the cell culture substrate of the first cell compartment is pitted or smooth;

[0052] - the cell culture substrate of the first cell compartment has a thickness between 5 nm and 200 pm;

[0053] - the cell culture substrate of the first cell compartment has a Young's modulus between 1kPa and 80kPa, preferably between 3kPa and 15kPa;

[0054] - 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;

[0055] - 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;

[0056] - 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; - the cell culture substrate of the first cell compartment includes fibroblasts and / or at least one immune cell, which makes it possible to reproduce the in vivo compositional variants of the lamina propria which incorporate such biological elements;

[0057] - at least part of the epithelial cells forming a monolayer or forming a multilayer is located within the cell culture substrate of the first cell compartment, which allows more physiological growth of the cells concerned and promotes their differentiation and the formation of layers that make up the epithelial tissue;

[0058] - the device includes muscle cells forming a layer located under the cell culture substrate of the first cell compartment or forming a layer intercalated within the cell culture substrate of the first cell compartment, which makes it possible to mimic the muscle mucosal layer present in vivo in target mucosa or mucosal-associated organ;

[0059] - the device includes an electrical stimulation system for muscle cells comprising electrodes arranged to electrically stimulate muscle cells, which allows the in vivo functioning of these muscle cells to be reproduced;

[0060] - the first cellular compartment comprises a biological sample of the mucous organ relating to said mucous membrane, said biological sample being placed on the biological structure, preferably said biological sample being of the microbiota;

[0061] - the first cellular compartment comprises a layer of synthetic or natural mucus covering at least partially the biological structure and / or the biological sample of said first cellular compartment;

[0062] - The device includes a third cellular compartment, located below the first cellular compartment, comprising endothelial cells and a porous membrane interposed between the first cellular compartment and the third cellular compartment to facilitate physiological exchanges between the first cellular compartment and the third cellular compartment:

[0063] - the endothelial cells of the third cellular compartment are distributed over all the walls of the third cellular compartment;

[0064] - the third cellular compartment comprises a cell culture substrate forming a surface on which the endothelial cells are distributed, said cell culture substrate of the third cellular compartment being configured to promote the growth or quiescence of the endothelial cells; - the endothelial cells of the third cellular compartment are derived from induced pluripotent stem cells, preferably from human induced pluripotent stem cells;

[0065] - the neurons of the second cellular compartment (40) are derived from induced pluripotent stem cells, preferably from human induced pluripotent stem cells;

[0066] - the porous membrane intercalated between the first cellular compartment and the third cellular compartment includes pores ranging from 400 nm to 100 pm;

[0067] - the second cellular compartment and / or the means forming the biological interface rest(s) on a porous membrane, the latter being distinct or being of one piece with the porous membrane intercalated between the first cellular compartment and the third cellular compartment.

[0068] 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 mucous membrane of a mucous membrane organ.

[0069] Advantageously, the stimulus is chosen from the list consisting of: a chemical stimulus, a biological stimulus, a mechanical stimulus, and a physical stimulus.

[0070] Advantageously, said mucosa is chosen from the list consisting of the mucosa of the mouth, the mucosa of the nasal passages, the mucosa of the respiratory tract, the mucosa of an organ of the digestive system and the mucosa of an organ of the genital tract.

[0071] Figures:

[0072] [Fig. 1]: Figure 1 is a schematic perspective representation of the device of the invention according to a first embodiment;

[0073] [Fig. 2]: Figure 2 is an exploded schematic representation of the device according to the first embodiment of the invention shown in Figure 1;

[0074] [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;

[0075] [Fig.4]: Figure 4 is a schematic top view representation of the device according to the first embodiment of the invention shown in Figure 1, of which an enlargement of the part illustrating the means forming biological interface, [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;

[0076] [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;

[0077] [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;

[0078] [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;

[0079] [Fig. 9]: Figure 9 is a schematic cross-sectional representation, along the section plane AA shown in Figure 4, of a device according to a second embodiment of the invention;

[0080] [Fig. 10]: Figure 10 is a schematic cross-sectional representation, according to the section plane AA shown in Figure 4, of the device shown in Figure 9 in which the biological elements have been schematically reproduced;

[0081] [Fig. 11]: Figure 11 shows two visualizations (Figures 11A and 11B) highlighting, by fluorescence, the cell cultures implemented in the device according to the invention. The second visualization (Figure 11B) highlights the innervation of the cell culture substrate of the first cell compartment;

[0082] [Fig. 12]: Figure 12 represents two visualizations (Figures 12A and 12B) highlighting the neurons, their neuronal connections (axons) and their neuronal terminals within the device according to the invention.

[0083] The present invention is illustrated in a non-limiting way by the following examples.

[0084] Device 1 according to the invention is intended to allow a reproduction as close as possible to the in vivo conditions of the mucous membrane of the mucous organ which is reproduced in the first cellular compartment 10. For reasons 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 remain evidently present.

[0085] To this end, the invention relates to a multi-compartment microfluidic device 1 comprising:

[0086] - a first cellular compartment 10 comprising a biological structure derived (103, 104) from the mucosa of a mucous organ comprising epithelial cells 100 of said mucosa, a cell culture substrate 11 forming a surface on which said biological structure (103, 104) is disposed and being configured to promote the growth or quiescence of the epithelial cells 100 of the biological structure (103, 104) and to allow, through itself, physiological exchanges with said epithelial cells 100,

[0087] - a second cellular compartment 40 comprising neurons 400 distributed on a cell culture substrate 41 comprising a plurality of electrodes 44 configured to measure the functional activity of the neurons 400, and

[0088] - at least one means 50 forming a biological interface to allow communication by neuronal connection 401 between the first cellular compartment 10 and the second cellular compartment 40.

[0089] As illustrated in particular in Figures 1 to 3B, the device 1 according to the invention has a casing-like shape delimiting a volume within which several distinct cellular compartments coexist, namely three distinct compartments (10, 20, 40) for the first embodiment of the invention (Figures 1 to 8) and two distinct cellular compartments (10, 40) for the second embodiment of the invention (Figures 9 and 10). This internal volume defined by the device 1 is delimited by the lid 2 and the base 3.

[0090] 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 of the digestive system, whose function is to be mimicked, are deposited on a cell culture substrate 11. These cells 100 can be cultured to form a monolayer 103 of epithelial cells 100 (Figures 6 and 8) or a multilayer of epithelial cells 100 (Figures 7 and 10). Alternatively, according to unshown variations of the two embodiments of the device 1 according to the invention, another biological structure derived from the target mucosa, for example, a spheroid, an organoid, or a tissue or expiry material, can be positioned. According to another unshown variation of the invention, at least a portion of the epithelial cells 100 of said biological structure can be contained within the cell culture substrate 11 itself.

[0091] The cell culture substrate 11 of this first cell compartment 10 is an important element of the invention since, by its nature, it allows for the mimicking of the composition and function of the lamina propria, a layer of loose connective tissue in the mucous membranes of mucous organs, whose role is crucial for reproducing the in vivo environment of these organs. To this end, this cell culture substrate 11 has mechanical properties that allow for control of the surface condition, porosity, viscosity, and roughness of the substrate. Furthermore, it is non-cytotoxic since it promotes the growth or quiescence of epithelial cells 100. In this respect, this cell culture substrate 11 can be loaded with collagen and / or elastin in concentrations ranging from 1 to 10 mg / ml (preferably 3 to 6 mg / ml).To further mimic the composition and function of the lamina propria, the cell culture substrate 11 may also include fibroblasts and / or immune cells. As illustrated throughout the figures, this cell culture substrate 11 may have a smooth surface, thus facilitating the deposition of the derived biological structure (103, 104). Alternatively, according to a variant not shown, the cell culture substrate 11 may include a plurality of protrusions (micro- or macro-modeled) projecting from its surface. This makes it possible to reproduce shapes mimicking the villi of the lamina propria. Alternatively, the surface of this cell culture substrate 11 may also be pitted.

[0092] To personalize the target mucosa to be reproduced in the first compartment 10, a biological sample 101 (such as microbiota or saliva) from the associated mucosal organ, taken from an individual, can be deposited onto the epithelial cells 100 of the derived biological system. This biological sample 101 can be positioned directly on the epithelial cells 100 or placed on a layer of synthetic or natural mucus 102, interposed between the layer of epithelial cells 100 and the biological sample 101 (Figure 8), in order to protect the epithelial cells 100 from aggressions from elements of the biological sample 101, such as pathogens and irritants present in the microbiota or saliva.

[0093] 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.

[0094] The second 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 second cellular compartment 40 and extend their respective neuronal terminals 402 to 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 makes it possible to digitize the behavior of the epithelial cells 100, mimicking the target mucosa and its function, in response to said stimuli.

[0095] The means 50, forming a biological interface to enable communication via neuronal connection 401 between the first cellular compartment 10 and the second 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.

[0096] Advantageously, the device 1 according to the invention can be provided with a third cell compartment 20. This third cell compartment 20, as shown in Figures 1 to 8, illustrating a first embodiment of the device 1 according to the invention, 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 third 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 third cellular compartment 20 is located below the first cellular compartment 10 and is separated from it by the porous membrane 30, thus allowing the epithelial cells 100 present in the first compartment 10 to benefit from the effects of vascularization, generated by the endothelial cells 200 organized into a blood vessel, similar to the effects present in vivo.

[0097] The porous membrane 30, as shown in Figures 1 to 8, illustrating the first embodiment of the device 1 according to the invention, is a thin membrane interposed between the first cell compartment 10 and the third cell compartment 20 to facilitate physiological exchanges between these two compartments and their respective cells and other biological elements. According to a first variant of this 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 the cell culture substrate 41 of the second cell compartment 40, a substrate incorporating the electrodes 44.This membrane 30 also forms the substrate on which the axons of neurons 400 extend through the plurality of microchannels 51 thus forming the neuronal connections 401 of the means 50 forming the biological interface allowing communication by neuronal connection between the first cellular compartment 10 and the second cellular compartment 40.

[0098] According to the variant shown in Figure 3A, the porous membrane 30 further comprises additional electrodes 45 located at the interface between the first cell compartment 10 and the third 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 third cell compartment 20 and the study of the cellular response to such a stimulus. By way of an advantageous and non-limiting example of this variant of the invention, the first cell compartment can incorporate muscle cells (not shown) from the target mucosa, forming a layer located beneath the cell culture substrate 11 of the first cell compartment 10 or forming a layer intercalated within the cell culture substrate 11.Therefore, the additional electrodes 45 form an electrical stimulation system for these muscle cells, allowing them to be electrically stimulated, thus reproducing the muscle contractions that these cells can experience in vivo.

[0099] According to the variant of the invention shown in Figure 3B, the device 1 comprises a porous membrane 30 not forming entirely the cell culture substrate 41 of the second cell compartment 40. This device comprises a separate membrane 31 forming the part of said cell culture substrate 41 incorporating the plurality of electrodes 44. This independent membrane 31 further has an opening 32 located at the interface between the first cell compartment 10 and the third cell compartment 20, so as not to impact the physiological exchanges between these two compartments.

[0100] Device 1 according to the invention works as follows.

[0101] A biological structure derived from the mucosa of a mucous organ is positioned on the surface of the cell culture substrate 11 of the first cell compartment 10. This biological structure comprises epithelial cells 100 from the mucosa whose function and environment are to be mimicked and reproduced. For example, squamous, salivary, or olfactory epithelial cells 100, forming a monolayer 103 of epithelial cells 100 or a multilayer 104 of epithelial cells 100, are positioned. After these cells have colonized the entire surface of the cell culture substrate 11 of the first cell compartment 10, a biological sample 101 from the mucous organ corresponding to the target mucosa, taken from an individual, can be 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 mucosal organ associated with the patient's target mucosa, thus enabling the biological system reproduced within the first cellular compartment 10 to be functionalized while being personalized to the individual from whom said biological sample 101 was taken. Alternatively, depending on the intended purpose, said biological sample 101 may be a biological fluid, such as saliva. Alternatively, depending on the stimuli to be tested, it is possible to place no additional biological element on said epithelial cells 100.

[0102] In parallel, neurons 400 (e.g., sensory neurons) are introduced and cultured within the second cell compartment 40, with the aim of innervating the target mucosa 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 allow neuronal communication between the first cell compartment 10 and the second cell compartment 40 (Figures 11A to 12B). As previously mentioned, these neurons 400 are distributed on a cell culture substrate 41 comprising a plurality of electrodes.

[0103] 44 allowing the measurement of the functional activity of neurons 400.

[0104] In the advantageous embodiment of the invention shown in Figures 1 to 8, in which the device 1 includes a third cellular compartment 20, endothelial cells 200 are introduced and cultured within the third cellular compartment 20 with the aim of colonizing 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 mucosa reproduced in the first cellular compartment 10.

[0105] 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).

[0106] 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 composition, or a biological stimulus, such as a pathogen (bacteria or virus that trigger an immune response) or a new nutrient, by applying it directly to the biological sample 101 or to the epithelial cells 100 of the mucosa-derived biological structure that one wishes to reproduce. Such a stimulus causes reactions within the first cellular compartment 10, particularly within the epithelial cells 100. These reactions result in changes to the electrical activity of the neuronal terminals 402, which is recorded by the electrodes 44 and subsequently analyzed.Alternatively or cumulatively, the application of this chemical or biological stimulus can be indirect, by applying it within the third cellular compartment 20, which can then be transmitted to the epithelial cells 100 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.

[0107] 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 epithelial cells 100 of the biological structure derived from the mucosa that one wishes to reproduce. It is thus possible to evaluate the reactions within the first cellular compartment 10, in particular those of the epithelial cells 100, in response to such a stimulus. Such an 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.

[0108] Finally, Figures 11 and 12 show fluorescence visualizations of the cell populations within the device 1 according to the invention. These visualizations notably demonstrate the innervation capacity of the neuronal cells 400 of the second cellular compartment 40, via pluralitys of microchannels 51 of the medium 50 forming the biological interface between the first cellular compartment 10 and the second cellular compartment 40, through which the neuronal connections 401 of the neurons 400 extend. In Figure 11B, where the epithelial cells 100 of the mucosa have been intentionally cultured so as not to cover the entire surface of the cell culture substrate 11 of the first cellular 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 cellular compartment 10.

[0109] List of references

[0110] 1: Multi-compartment microfluidic device

[0111] 2: Lid of the multi-compartment microfluidic device

[0112] 3: Base of the multi-compartment microfluidic device

[0113] 10: first cellular compartment

[0114] 11: Cell culture substrate

[0115] 20: third cellular compartment

[0116] 21: Entry of the third cellular compartment

[0117] 22: exit from the third cellular compartment

[0118] 30: porous membrane

[0119] 31: distinct membrane incorporating the substrate of the second cellular compartment

[0120] 40: second cellular compartment

[0121] 41: Cell culture substrate of the second cell compartment covered with electrodes

[0122] 42: entrance to the second cellular compartment

[0123] 43: exit from the second cell compartment

[0124] 44: electrode(s)

[0125] 45: Additional electrode(s)

[0126] 50: medium forming the biological interface between the first cellular compartment and the second cellular compartment

[0127] 51: Fluidic microchannel

[0128] 100: epithelial cells of the target mucosa

[0129] 101: biological sample of the mucous membrane organ related to the target mucosa taken from an individual

[0130] 102: mucus layer

[0131] 103: monolayer of epithelial cells

[0132] 104: multilayer of epithelial cells

[0133] 200: endothelial cell(s)

[0134] 400: neuron(s)

[0135] 401: Neural connection(s)

[0136] 402: neuronal terminal(s)

Claims

Tl DEMANDS 1. Multi-compartment microfluidic device (1) comprising: a first cellular compartment (10) comprising a biological structure (103; 104) derived from a mucous membrane of a mucous membrane organ comprising epithelial cells (100) of said mucous membrane, a cell culture substrate (11) forming a surface on which said biological structure (103; 104) is disposed and being configured to promote the growth or quiescence of the epithelial cells (100) of the biological structure (103; 104) and to permit, through itself, physiological exchanges with said epithelial cells (100), a second cellular compartment (40) comprising neurons (400) distributed on a cell culture 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 permit communication by neuronal connection (401) between the first cellular compartment (10) and the second cellular compartment (40).

2. Device (1) according to the preceding claim, wherein the biological structure (103; 104) derived from a mucous membrane of a mucous organ is selected from the list consisting of: epithelial cells (100) forming a monolayer (103), epithelial cells (100) forming a multilayer (104), a spheroid, an organoid or a tissue or an expiant.

3. Device (1) according to any one of the preceding claims, wherein epithelial cells (100) of the biological structure (103; 104) derived from a mucous membrane of a mucous organ cover the entire surface of the cell culture substrate (11).

4. Device (1) according to any one of the preceding claims, wherein the cell culture substrate (11) of the first cell compartment (10) has a Young's modulus between 1kPa and 80kPa.

5. Device (1) according to any one of the preceding claims, wherein the cell culture substrate (11) of the first cell compartment (10) is further loaded with collagen, elastin, proteoglycans, glycosaminoglycans and / or structural glycoproteins.

6. Device (1) according to any one of the preceding claims, wherein the first cellular compartment (10) comprises a biological sample (101) of the mucous organ relating to said mucous, said biological sample (101) being disposed on the biological structure, preferably said biological sample (101) being of the microbiota.

7. Device (1) according to any one of the preceding claims, comprising a third cell compartment (20), located below the first cell compartment (10), comprising endothelial cells (200), and a porous membrane (30) interposed between the first cell compartment (10) and the third cell compartment (20) to facilitate physiological exchanges between the first cell compartment (10) and the third cell compartment (20).

8. Device (1) according to the preceding claim, wherein the endothelial cells (200) of the third cellular compartment (20) are distributed over all the walls of the third cellular compartment (20).

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 mucous membrane of a mucous organ.

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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