Method for evaluating the effects of a stimulus on a reproduction of a target organ and multi-compartment organ-on-chip enabling the implementation thereof

Abstract

The present invention relates to a method for evaluating the effects of a stimulus on a biological model derived from a target organ and a multi-compartment microfluidic device (1) enabling the implementation of said method.

Description

[0001] TITLE: METHOD FOR EVALUATING THE EFFECTS OF A STIMULUS ON THE REPRODUCTION OF A TARGET ORGAN AND MULTICOMPARTMENTAL ORGAN-ON-CHIP ENABLING ITS IMPLEMENTATION. FIELD OF THE INVENTION

[0002] The present invention relates to the field of microfluidic devices that mimic organs (human or animal) at the microscopic scale. Such devices are also known as organ-on-a-chip. More specifically, the invention relates to an in vitro method for evaluating the effects of a stimulus on a biological structure derived from a target organ, and to a compartmentalized device for implementing this method. This method and device allow for adaptation according to the target organ whose function is to be reproduced in vitro for analysis and / or diagnosis, and for personalization to each individual.

[0003] STATE OF THE ART

[0004] 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.

[0005] 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.

[0006] A second disadvantage is that the cells used in current devices are generally too pure, in that they lack the essential supporting cells necessary for the proper functioning of other cells and therefore crucial when aiming to reproduce the function of a specific organ as accurately as possible. Conversely, the cells used sometimes exhibit significant heterogeneity, with no control over the cell types present or their organization. Here again, the most accurate possible reproduction of the target organ and its function appears impossible. Finally, the devices currently available on the market are generally specific to a single organ or organ type and do not allow for the implementation of a method for evaluating the effects of stimuli applied to a very wide range of target organs.Furthermore, these devices are generally built for the evaluation of a specific type of stimulus and therefore do not allow for the analysis of the effects of a wide and varied panel of stimuli.

[0007] Consequently, current devices and the processes they employ do not allow for the evaluation of the effects of a wide range of stimuli on the highly accurate reproduction of a target organ, this target organ being customizable for each individual chosen from a very wide selection of target organs according to their needs. Thus, current devices and the processes they employ do not allow for satisfactory specific conclusions to be drawn regarding a plurality of target organs.

[0008] DESCRIPTION OF THE INVENTION

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

[0010] An objective of the present invention is to provide a method and an associated compartmentalized microfluidic device of the organ-on-a-chip type, which allow both a very faithful reproduction of the target organ, in all its structural complexity, an optimized personalization of said organ for each individual and an adaptability to different target organs and / or stimuli whose impact on the target organ is to be evaluated.

[0011] Thus, the present invention relates to an in vitro method for evaluating the effects of a stimulus on a biological model derived from a target organ, said method comprising the following steps:

[0012] a) Cellular dissociation of a biological structure derived from said target organ, said biological structure comprising cells (100) forming a 3D structure (A), b) Culture of the dissociated cells (B),

[0013] c) Obtaining the biological model derived from said target organ (C),

[0014] d) Application of said stimulus to the biological model derived from said target organ (D), e) Recording of the functional activity of neurons present within said biological model and / or neurons of a first neuronal culture and / or neurons of a second neuronal culture, the nerve endings of the neurons present within said biological model and of the first neuronal culture forming a neuronal connection between the cells of said biological model and the neurons of the first neuronal culture, the nerve endings of the neurons of the first neuronal culture and of the neurons of the second neuronal culture forming a neuronal connection between the neurons of the first neuronal culture and the neurons of the second neuronal culture, obtaining a derived model of neuronal functional activity (E).

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

[0016] - a highly accurate and relevant reproduction of the structural complexity of the target organ, since a biological structure comprising all the cells involved in said target organ is dissociated and then cultured to obtain a derived biological model representative of said target organ, on which the chosen stimulus will be tested. Therefore, it is possible, through this dissociation of the biological structure, to obtain, in the most comprehensive way possible, each of the cell types that compose said target organ, including cell types that can be difficult, or even impossible, to reproduce using current classical differentiation protocols.

[0017] - a personalization of the biological model derived from the reproduced organ since it can be adapted to a type of individual chosen on whom the effects of the stimulus are to be evaluated, or even be made from a biological sample taken directly from the individual on whom the effects of the stimulus are to be evaluated (for example in the context of a personalized diagnosis or an efficacy test of a new drug or treatment),

[0018] - The method and associated device allow the effects of a broad panel of stimuli (e.g., nutrients, creams, medications, probiotics, etc.) to be measured on the biological model derived from the target organ; - The method and associated device also allow the study of the effects of the stimulus and / or the functioning of the network composed of the target organ, the peripheral nervous system (PNS), and the central nervous system (CNS).

[0019] - The process and associated device allow for a controlled and reproducible approach, avoiding the direct use of in vivo experiments,

[0020] - a generic, economical and standardized device since it allows the culture of dissociated cells from many types of biological structures (including many different types of biological samples and / or from different target organs).

[0021] In the context of the invention, "in vitro method for evaluating the effects of a stimulus on a biological model derived from a target organ" means an experimental method aimed at studying and quantifying the biological, physiological or pathological responses induced by the application of a specific stimulus on a biological model mimicking a target organ.The assessments carried out in this context can cover a wide range of objectives, including: diagnostic assessment to detect abnormalities or pathologies; pharmacological efficacy tests aimed at measuring the effect of therapeutic molecules or treatments on specific responses, such as the inhibition of inflammation or tissue regeneration; toxicity studies to determine the cytotoxic, genotoxic or immunotoxic effects of the compounds tested; modeling of pathologies by the in vitro reproduction of specific pathological conditions to study the progression of diseases or their underlying mechanisms; validation of medical devices to assess the impact of a device or material (such as an implant or prosthesis) on the target organ-derived biological model; and environmental studies to analyze the effects of external agents such as pollutants on the target organ-derived biological model.For the purposes of this invention, a "compartmentalized microfluidic device" is defined as a miniaturized device comprising one or more distinct compartments, each designed to house and culture cells, which may be of different cell types. Each compartment is designed to mimic a specific part of the organism, thus enabling the reproduction of biological interactions between various tissues or organs in a controlled environment. In the case of a multi-compartmentalized microfluidic device, the compartments are interconnected, for example, by microfluidic channels, allowing the transfer of fluids, nutrients, or biological signals, such as bioelectrical signals via neuronal connections, thereby simulating the 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 compartmentalized 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 reproducing 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 organ" refers to the specific organ of the organism that is to be reproduced in the first compartment of the compartmentalized 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. The invention is relevant to all organs of the human body, and particularly to the intestines, brain, liver, kidneys, and lungs. Organs with mucous membranes, which are covered by a layer of epithelial cells that secrete mucus and play a key role in exchange and protection against the external environment, are also of interest. These organs include, in particular, the intestines, colon, lungs, skin, stomach, nose, mouth, esophagus, vagina, and urinary tract.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.

[0024] In the context of the invention, a "target organ-derived biological structure" is understood to be an entity formed from cells of said target organ, intended to be dissociated and cultured to obtain a biological model derived from that same organ, thus reproducing that organ in a microfluidic device such as that according to the invention. This entity may originate from a biological sample of the target organ, such as a tissue sample or an expiry. It may also consist of more complex biological assemblies, such as cell aggregates, spheroids, or organoids, enabling the reproduction of advanced functional and structural features of the target organ. This structure may be generated, at least partially, from induced pluripotent stem cells (iPSCs), derived and reprogrammed to mimic the specific characteristics of the target organ to be reproduced.

[0025] In the context of this invention, "cellular dissociation of a biological structure derived from said target organ" means a process for disaggregating a biological structure comprising cells organized in three dimensions in order to obtain an individual cell solution. This biological structure, derived from a target organ, may include, but is not limited to, spheroids, organoids, expiants, or any other cellular aggregate mimicking the architecture and cellular interactions of the organ of origin. Cell dissociation is carried out by appropriate mechanical, enzymatic, and / or chemical means to preserve the integrity and viability of the cells obtained. This step allows for the isolation of a heterogeneous population comprising all the cell types present in the initial biological structure, so as to maintain the cellular diversity representative of the target organ.The cell solution thus obtained can be used to establish a new cell culture intended to reconstitute a biological model derived from said target organ in order to evaluate the effects of a given stimulus.

[0026] For the purposes of this invention, a "cell aggregate" is defined as a three-dimensional structure composed of cells adhering to one another, formed by self-organization or by specific culture methods that promote cell interaction and cohesion. This aggregate may result from the culture of cells of the same type or of different cell types, thus allowing for the reproduction, at least partially, of the tissue organization and intercellular interactions present in a given organ or tissue. Cell aggregates may be composed of, but not limited to, epithelial, fibroblastic, neuronal, muscle, immune, or endothelial cells. The cell aggregate may also serve as a starting point for the formation of spheroids or organoids, thereby extending cell differentiation and maturation in culture.In the context of this invention, a "spheroid" is defined as a three-dimensional structure composed of an aggregate of cells, primarily epithelial cells, 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 or pathogens.

[0027] In the context of this invention, an "organoid" is defined as a three-dimensional structure derived from stem cells or primary cells, which functionally and structurally reproduces certain characteristics of a target organ. An organoid reflects several aspects of the architecture and specific functions of the target organ and, as such, constitutes a robust biomimetic model.

[0028] In the context of this invention, "tissue" means an organized assembly of several types of cells from a target organ, cultured in the laboratory from biological samples. This tissue reproduces the interactions between the different cells of the target organ and is used, in particular, to study the physiology and pathologies of said target organ in a controlled environment.

[0029] For the purposes of this invention, an "expiant" is defined as 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 tissue derived from the target organ that one seeks to reproduce.

[0030] In the context of the invention, a "target organ-derived biological model" is defined as an entity obtained by culturing dissociated cells from a biological structure derived from said target organ, to which the stimulus whose effects are to be evaluated is applied. These biological models may take the form of cells forming a monolayer or a multilayer. These entities are used to model the biological functions and interactions specific to target organs in an experimental or therapeutic context. These cells may be any cell type necessary for the reproduction of said target organ. By way of non-exhaustive example, the cells forming target organ-derived biological models may be chosen from: epithelial cells (squamous, cuboidal, columnar, ciliated, glandular, endothelial, mesothelial, mucous, olfactory, gustatory, enterocytes, goblet cells, and Panet cells),connective and supporting cells (fibroblasts, myofibroblasts, adipocytes, chondrocytes, osteoblasts, osteocytes, osteoclasts, tenocytes, and pericytes), muscle cells (skeletal, smooth, myocardial, and satellite), nerve cells (sensory neurons, motor neurons, interneurons, glutamatergic, GABAergic, dopaminergic, serotonergic, and cholinergic neurons), glial cells (astrocytes, oligodendrocytes, Schwann cells, microglia, and ependymal cells), blood and immune cells (erythrocytes, leukocytes including neutrophils, B, T, and NK lymphocytes, monocytes, eosinophils, basophils, macrophages, dendritic cells, and platelets), and endocrine cells (thyrocytes, adrenal cells, pancreatic alpha, beta, and delta cells, Leydig cells, and...) Sertoli and granulosa cells), reproductive cells (oocytes, spermatozoa, follicular and luteal cells),as well as specialized sensory cells (photoreceptors, auditory and vestibular hair cells, Merkel cells). These models thus allow the study of various biological processes such as tissue regeneration, responses to external stimuli, and complex cell interactions.

[0031] In the context of the invention, "monolayer" means a cellular structure composed of a single layer of cells from the target organ.

[0032] In the context of the invention, "multilayer" means a cellular structure composed of several superimposed layers of cells, allowing the architectural and functional complexity of the biological tissues of the targeted organ to be reproduced.

[0033] For the purposes of this invention, "neurons" are defined as nerve cells specialized in transmitting electrical and chemical signals within the nervous system. Generally, neurons are composed of three main parts: the soma, or cell body, which contains the nucleus and constitutes the cell's metabolic center; the dendrites, branching extensions that receive signals from other cells; and the axon, a single extension often surrounded by a myelin sheath, which enables the rapid transmission of electrical signals to other cells or muscles. These cells play a crucial role in communication and the coordination of biological functions by transmitting signals between different parts of the body and enabling the body's sensory and motor responses.Within the framework of the invention, particular interest is given to the types of neurons that can be integrated into one of the cellular compartments of the device according to the invention and that can develop in such a way as to innervate, via their respective nerve 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; autonomic neurons, which control involuntary functions such as heart rate and digestion by relaying signals to internal organs; and glutamatergic neurons, which release glutamate as the main excitatory neurotransmitter in the central nervous system, playing a key role in processes such as synaptic plasticity, learning, and memory.

[0034] In the context of this invention, "functional neuronal activity" refers to the emission and propagation of a nerve impulse in the form of electrical signals and / or neurotransmitter secretions. Functional activity is captured using appropriate technical means, including, but not limited to: multi-electrode arrays (MEAs) or high-density multi-electrode arrays (HDMEAs), enabling the detection of action potentials and their propagation; functional imaging, based on calcium indicators or voltage-sensitive probes, to visualize variations in neuronal activity in real time; and electrophysiological methods, such as patch-clamp or local field recordings, for detailed characterization of the electrical properties of neurons.

[0035] In the context of this invention, a "derived model of neuronal functional activity" is understood to be an experimental representation that allows for the reproduction and study of the functional activity of neurons, defined as the emission and propagation of a nerve impulse in the form of electrical signals and / or neurotransmitter secretions. This model is based on the recording and analysis of neuronal activity within the biological structure, if it directly contains neurons, and / or of independent neuronal cultures, which may include a first neuronal culture and a second neuronal culture, in response to the application of a stimulus to said biological structure.

[0036] In the context of this invention, a "reference model of neuronal functional activity" is understood to mean an experimental representation used to establish the characteristics of baseline neuronal activity in the absence of an external stimulus applied to the biological structure in question, or a model based on existing data relating to the target organ from which the biological structure is derived. This model allows observation of the functional activity of neurons in a basal state, before any modification induced by external stimuli, and serves as a point of comparison for evaluating the effects of these stimuli. This reference model thus provides a basis for comparison for evaluating the functional responses of neurons under different experimental conditions.

[0037] For the purposes of this invention, a "cell culture substrate" is defined as a support, generally solid, that allows for the adhesion, growth, and proliferation of cultured cells. The substrate may 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 a supporting layer (such as the lamina propria in a mucosal organ), thus ensuring a physicochemical environment close to the physiological conditions of the target organ.For the purposes of this invention, "individual" means any living organism, human or animal, from which biological samples can be taken to personalize and functionalize cells mimicking the target organ in the device. Humans are the preferred source for collecting these samples, but animals, particularly in preclinical models or comparative studies, can also be used. These individuals serve as a biological source for adapting in vitro cell models to the specific physiological and pathological characteristics of each organism.In the context of this invention, a "means forming a biological interface to enable communication via neuronal connections between 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 the neuronal connections of the neuronal cells present in one or both of said cellular compartments, enabling the transfer of biological information in the form of electrical or chemical signals between these two compartments.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 110. 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.

[0038] For the purposes of this invention, "induced pluripotent stem cells" are defined as cells derived from adult somatic cells that have been reprogrammed to regain a pluripotent state, similar to that of embryonic stem cells. These cells, commonly called iPSCs (induced pluripotent stem cells), have the capacity to differentiate into virtually any cell type in the body, including neuronal, cardiac, hepatic, or epithelial cells. Human induced pluripotent stem cells (hiPSCs) can be derived from adult human cells originating, for example, from organs of the digestive system, such as cells from the intestine, stomach, or colon.

[0039] 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.

[0040] In the context of this invention, "evaluation of the effects of said stimulus on the biological model derived from said target organ for diagnostic purposes" means a method for characterizing or identifying specific biological markers within a biological model derived from a target organ, in response to an applied stimulus. These markers may include, but are not limited to, variations in gene expression, protein production, morphological changes, or functional alterations such as neuronal or contractile activity. The primary objective of this evaluation, for example, is to enable the detection, identification, or confirmation of a pathological condition, a functional abnormality, or a specific susceptibility to a disease for diagnostic or clinical monitoring purposes.

[0041] In the context of this invention, "evaluation of the effects of said stimulus on the biological model derived from said target organ for efficacy testing purposes" refers to a method for measuring and quantifying the beneficial or therapeutic effects of an applied stimulus, such as a candidate molecule, a drug, a biomaterial, or a physical intervention, on a biological model derived from a target organ. The evaluation may include, but is not limited to, improvements in the model's biological functions, tissue repair, reduction of stress or inflammation markers, or improvements in functional parameters such as secretion, absorption, or contractile activity. This method can be used, in particular, to validate the efficacy of a product or intervention for its development or clinical application.

[0042] In the context of this invention, "evaluation of the effects of said stimulus on the biological model derived from said target organ for toxicity testing purposes" means a method for detecting, measuring, and analyzing the deleterious or undesirable effects of a stimulus applied to a biological model derived from a target organ. These effects may include, but are not limited to, morphological alterations, disruption of physiological functions, cell death (apoptosis or necrosis), DNA damage, or metabolic disturbances. This method can be used to assess the safety and risks associated with a chemical, therapeutic molecule, biomaterial, or environmental substance prior to its clinical or industrial use.

[0043] Preferably, the present invention relates to an in vitro method for evaluating the effects of a stimulus on a biological model derived from a target organ having the following technical characteristics, taken alone or in combination:

[0044] - a step (F), prior to step (D), of recording the functional activity of the neurons present within said biological model and / or the neurons of a first neuronal culture and / or the neurons of a second neuronal culture, and of obtaining a reference model of neuronal functional activity;

[0045] - a step (G) of comparison between the reference model of neuronal functional activity and the derived model of neuronal functional activity;

[0046] - step (A) includes the use of a chemical compound and / or a biological compound to destroy the intercellular bonds of the biological structure while preserving the integrity of the cells and their function;

[0047] - step (B) further includes culturing said first neuronal culture so that the nerve endings of the neurons of this first neuronal culture innervate the cells of the biological model;

[0048] - step (B) further includes culturing said second neuronal culture so that the nerve endings of the neurons of this second neuronal culture innervate the neurons of the first neuronal culture;

[0049] - the cells of the biological model form a monolayer or a multilayer, said cells preferably being derived from induced pluripotent stem cells, preferably from human induced pluripotent stem cells;

[0050] - the biological structure derived from said target organ is chosen from the list consisting of: a cell aggregate, a spheroid, an organoid, a tissue and an expiant, the tissue and / or the explant preferably being taken from an individual;

[0051] - the first neuronal culture comprises sensory neurons, said sensory neurons preferably being derived from induced pluripotent stem cells, preferably from human induced pluripotent stem cells; - the second neuronal culture comprises glutamatergic neurons, said glutamatergic neurons preferably being derived from induced pluripotent stem cells, preferably from human induced pluripotent stem cells;

[0052] - the dissociated cells from the biological structure are cultured in a first cellular compartment, the first neuronal culture is cultured in a second cellular compartment and the second neuronal culture is cultured in a third cellular compartment;

[0053] - the neuronal terminals of the first neuronal culture extend within a first means forming a biological interface to allow communication by neuronal connection between the first cellular compartment and the second cellular compartment and / or in which the neuronal terminals of the second neuronal culture extend within a second means forming a biological interface to allow communication by neuronal connection between the second cellular compartment and the third cellular compartment;

[0054] - the recording of the functional activity of neurons is carried out via a plurality of electrodes arranged under the culture substrate of the first cellular compartment, under the culture substrate of the second cellular compartment and / or under the culture substrate of the third cellular compartment;

[0055] - said stimulus is chosen from the list consisting of: a chemical stimulus, a biological stimulus, a mechanical stimulus and a physical stimulus;

[0056] - said stimulus is chosen from the list consisting of: a drug, a molecule, a cream, a probiotic, added to the biological structure;

[0057] - the evaluation of the effects of said stimulus on the biological model derived from said target organ is for diagnostic purposes, for efficacy testing purposes, for toxicity testing purposes; or

[0058] - the said target organ is chosen from the list consisting of: the intestine, the brain, the liver, the kidney and the lung.

[0059] The invention also relates to a multi-compartment microfluidic device for implementing the process according to any one of the preceding variants of the invention, said device comprising:

[0060] - a first cellular compartment comprising the biological model derived from said target organ, a first cell culture substrate forming a surface on which said biological model is disposed and being configured to promote the growth or quiescence of the cells of said biological model,

[0061] - a second cellular compartment comprising the first neuronal culture, a second cell culture substrate forming a surface on which said first neuronal culture is disposed and configured to promote the growth or quiescence of neurons in said first neuronal culture, and

[0062] - at least one first means forming a biological interface to allow communication by neuronal connection between the first cellular compartment and the second cellular compartment.

[0063] Advantageously, the device according to the invention has the following technical characteristics, taken alone or in combination:

[0064] - a third cellular compartment comprising the second neuronal culture, a third cell culture substrate forming a surface on which said second neuronal culture is disposed and being configured to promote the growth or quiescence of neurons of said second neuronal culture, and at least a second means forming a biological interface to enable communication by neuronal connection between the second cellular compartment and the third cellular compartment;

[0065] - a fourth cellular compartment, located below the first cellular compartment, comprising endothelial cells, and a porous membrane interposed between the first cellular compartment and the fourth cellular compartment to facilitate physiological exchanges between the first cellular compartment and the third fourth cellular compartment; - the endothelial cells are distributed over all the walls of the fourth cellular compartment;

[0066] - the fourth cellular compartment comprises a cell culture substrate forming a surface on which endothelial cells are distributed, said cell culture substrate of the fourth cellular compartment being configured to promote the growth or quiescence of endothelial cells;

[0067] - The endothelial cells of the fourth cellular compartment are derived from induced pluripotent stem cells, preferably human induced pluripotent stem cells; - The porous membrane intercalated between the first and fourth cellular compartments comprises pores ranging from 400 nm to 100 pm. Figures:

[0068] [Fig. 1]: Figure 1 represents a flowchart illustrating the steps of the process of the invention according to a first embodiment;

[0069] [Fig. 2]: Figure 2 represents a flowchart illustrating the steps of the process of the invention according to a variant of the first embodiment;

[0070] [Fig. 3]: Figure 3 is a schematic perspective representation of the device of the invention according to a first embodiment;

[0071] [Fig.4]: Figure 4 is an exploded schematic representation of the device according to the first embodiment of the invention shown in Figure 3;

[0072] [Fig. 5]: Figure 5 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 3;

[0073] [Fig. 6]: Figure 6 is a schematic top view representation of the device according to the first embodiment of the invention shown in Figure 3, of which an enlargement of the part illustrating the means forming biological interface;

[0074] [Fig. 7]: Figure 7 is a schematic cross-sectional representation, along the section plane AA shown in Figure 6, of the device according to the first embodiment of the invention shown in Figure 3;

[0075] [Fig. 8]: Figure 8 is a schematic cross-sectional representation, along the section plane AA shown in Figure 6, of the device according to the first embodiment of the invention shown in Figure 3, in which the biological elements have been reproduced schematically;

[0076] [Fig. 9]: Figure 9 is a schematic cross-sectional representation, along the section plane AA shown in Figure 6, of a third variant embodiment of the device according to the first embodiment of the invention;

[0077] [Fig. 10]: Figure 10 is a schematic cross-sectional representation, along the section plane AA shown in Figure 6, of a fourth variant of the device according to the first embodiment of the invention; [Fig. 11]: Figure 11 is a schematic cross-sectional representation, along the section plane AA shown in Figure 6, of a device according to a second embodiment of the invention;

[0078] [Fig. 12]: Figure 12 is a schematic cross-sectional representation, according to the section plane AA shown in Figure 6, of the device shown in Figure 11 in which the biological elements have been schematically reproduced;

[0079] [Fig. 13]: Figure 13 is a schematic perspective representation, along the section plane AA shown in Figure 6, of the device of the invention according to a third embodiment;

[0080] [Fig. 14]: Figure 14 illustrates a pool of four cortical organoids (A) prior to the dissociation step, said pool forming the biological structure derived from said target organ, in this case the brain, and the viability of the different dissociated cells, forming the biological model derived from said target organ, even after their seeding on multi-compartment microfluidic devices for the purpose of evaluating the effects of a stimulus on said biological model obtained.

[0081] The present invention is illustrated in a non-limiting way by the following examples.

[0082] The process according to the invention is intended to allow both a very faithful reproduction of a target organ, in all its structural complexity, an optimized personalization of said organ for each individual and an adaptability to different target organs and / or stimuli whose impact on the target organ is to be evaluated.

[0083] To this end, the invention relates to an in vitro method for evaluating the effects of a stimulus on a biological model derived from a target organ, said method comprising the following steps shown in Figure 1:

[0084] a) Cellular dissociation of a biological structure derived from said target organ, said biological structure comprising cells forming a 3D structure (A),

[0085] b) Culture of dissociated cells (B),

[0086] c) Obtaining the biological model derived from said target organ (C),

[0087] d) Application of said stimulus to the biological model derived from said target organ (D), e) Recording of the functional activity of neurons present within said biological model and / or neurons of a first neuronal culture and / or neurons of a second neuronal culture, the nerve endings of the neurons present within said biological model and of the first neuronal culture forming a neuronal connection between the cells of said biological model and the neurons of the first neuronal culture, the nerve endings of the neurons of the first neuronal culture and of the neurons of the second neuronal culture forming a neuronal connection between the neurons of the first neuronal culture and the neurons of the second neuronal culture, obtaining a derived model of neuronal functional activity (E).

[0088] During step A of the process according to the invention, a complex biological structure is undergoing cellular dissociation. This structure may be a complex biological assembly, such as cell aggregates, spheroids, organoids, or a biological sample from the target organ, such as a tissue sample or an expiry. This biological structure incorporates a multitude of cells specific to the target organ on which a stimulus is to be tested and its effects evaluated. To achieve cellular dissociation of the biological structure, a chemical and / or biological compound may be used that disrupts the intercellular bonds of the biological structure while preserving the integrity and function of the cells.

[0089] Following this cell dissociation, we obtain a solution comprising functional cells that represent all the cell types present in the initial biological structure.

[0090] During step B, the dissociated cells are cultured until a biological model derived from the same organ as the initial biological structure is obtained (step C). This biological model can take the form of cells forming a monolayer or a multilayer.

[0091] In parallel with the culturing of the dissociated cells 100, a first neuronal culture and a second neuronal culture can be cultured such that the nerve endings 402 of the neurons 400 of the first neuronal culture innervate the dissociated cells 100 intended to form the biological model and / or the second neuronal culture, and that the nerve endings 602 of the neurons 600 of this second neuronal culture innervate the neurons 400 of the first neuronal culture. Culturing the first neuronal culture is particularly necessary when the initial biological structure does not include neurons. Advantageously, the neurons 400 of the first neuronal culture include sensory neurons 400, which allows for the mimicry of the functioning of the peripheral nervous system on the target organ.The 600 neurons forming the second neuronal culture include 600 glutamatergic neurons which allow the functioning of the central nervous system to be mimicked on the peripheral nervous system and, ultimately, on the target organ.

[0092] During step D of the process according to the invention, a predetermined stimulus is applied directly or indirectly to the biological model derived from the target organ obtained in step C. This stimulus may be a chemical stimulus, such as a new drug candidate whose toxicity or reparative effects on a specific organ one wishes to evaluate, for example. It may also be a pathogen whose evolution and impact one wishes to observe within a specific biological environment of a target organ. During step E, the final step of the process according to the first embodiment of the invention as illustrated in Figure 1, the functional activity of neurons present within said biological model and / or neurons present within a cell culture separate from said biological model is recorded.This separate cell culture can be the first neuronal culture, whose nerve endings form a neuronal connection with the cells of the biological model. Alternatively, it can consist of neurons belonging to the second neuronal culture, which comprises neurons whose nerve endings form a neuronal connection with the neurons of the first neuronal culture. By recording the functional activity of all these neurons, a derived model of neuronal functional activity is obtained, revealing the effects of the tested stimulus on the biological model of the target organ.

[0093] Advantageously, as illustrated in Figure 2, the method according to the invention includes an additional step (Step F), preliminary to Step D, which consists of recording the functional activity of neurons present within said biological model and / or within the first neuronal culture and / or the second neuronal culture, in order to obtain a reference model of neuronal functional activity. In other words, this reference model represents the neuronal functional activity of said biological model derived from the target organ in the absence of an external stimulus. The reference model obtained in Step F can then be compared, during the additional step G, with the derived model of neuronal functional activity obtained in Step E, in order to evaluate the effects of said stimulus on the biological model and thus draw conclusions about the effects of said stimulus on the target organ from which the tested biological model is derived.

[0094] Example of a completed project

[0095] As illustrated in Figure 14A, the biological structure derived from said target organ can be a pool of several organoids (S1-S4) whose dissociation makes it possible to obtain a biological model derived from said target organ comprising all the cellular diversity specific to the target organ (in this example the target organ being the brain).

[0096] To achieve this, the following dissociation method, implemented with a STEMCELL™ Technologies kit, was used. It should be noted that this example is in no way limiting to the invention, as the inventors have demonstrated the viability of the cells constituting said biological models using other dissociation methods known to those skilled in the art, including those using, for example, a Miltenyi kit (dissociation using the GentleMACS system or manual dissociation without recourse to said system), as well as for other target organs. The following example presents the obtaining of a biological model derived from the brain, this organ being characterized by one of the most complex cellular variability (neurons, neural progenitors, astrocytes, etc.).Thus, maintaining the cellular viability of all cells originating from this biological structure demonstrates in a particularly convincing way the applicability of the process according to the invention to all target organs exhibiting less cellular complexity.

[0097] 1- Dissociation of organoids with STEMCELL™ Technologies kit. The four organoids S1-S4 were transferred into a 24-well plate well;

[0098] 2- The culture medium was removed, then a PBS wash was carried out before complete removal of the PBS;

[0099] 3- A volume of 500 pL of dissociation solution (solution initially prepared from a dilution of Papain Stock Solution and DNase I in HBSS to obtain the following final concentrations: Papain: 30 units / mL and DNase I: 125 units / mL) was added to the well;

[0100] 4- The sample was incubated at 37 °C for 30 minutes on an orbital shaker set at 80 rpm;

[0101] 5- An initial mechanical dissociation was carried out using a 1 mL pipette by 5 to 6 back-and-forth movements, in order to obtain a suspension of cell aggregates; 6- A second incubation was carried out at 37 °C for 10 minutes on an orbital shaker at 80 rpm;

[0102] 7- The final dissociation of the organoids was carried out using a 1 mL pipette by 5 to 6 back-and-forth movements, allowing a suspension of individual cells to be obtained;

[0103] 8- The cell suspension was transferred into a 15 mL Falcon tube containing 1.5 mL of ovomucoid protease inhibitor solution (trypsin inhibitor - solution obtained from the resuspension of trypsin inhibitor powder in HBSS to a final concentration of 10 mg / mL);

[0104] 9- The suspension was centrifuged for 5 minutes at 300 g;

[0105] 10- The supernatant has been removed;

[0106] 11- The cell pellet was resuspended in 1 mL of culture medium;

[0107] 12- A cell count was performed using a Malassez cell after staining with Trypan blue (1:1 mixture with a 0.4% Trypan blue solution);

[0108] 13- The cell suspension was filtered to remove debris and aggregates using a 40 µm cell sieve;

[0109] 14- A volume of 1 mL of PBS was added to rinse the cell sieve, leading to a final volume of 2 mL of cell suspension;

[0110] 15- A second count on a Malassez cell was carried out in order to determine the final cell concentration and to verify the elimination of aggregates;

[0111] 16- The suspension was centrifuged for 5 minutes at 300 g;

[0112] 17- The cells were resuspended in an appropriate volume of culture medium.

[0113] Cell counting revealed equivalent cell numbers and diversity after organoid dissociation, with a very low cell death rate. Seeding of the dissociated cells (forming the biological model)

[0114] 1- The inlets and outlets of the multi-compartment microfluidic device are checked to ensure that they are not dried out. If this is the case, 10 pL of seeding medium are added;

[0115] 2- The loading of the cells was carried out by placing the pipette cone upstream and in the axis of the channel to be seeded, the addition being carried out in one go and without jerking in order to ensure a homogeneous distribution of the cells in the active zone; 3- The device was incubated at 37 °C under an atmosphere containing 5 % CO2 for 1 hour, before the addition of the culture medium.

[0116] Fixation of dissociated cells and immunofluorescent labeling

[0117] 1- The cells were fixed in the microfluidic device as well as on coverslips placed in 24-well plates, using a 4% paraformaldehyde (PFA) solution for 20 minutes;

[0118] 2- Three washes with PBS were carried out, then the samples were kept at 4 °C in PBS until the immunofluorescent labels were carried out;

[0119] 3- Immunofluorescent labeling was performed in the microfluidic device and on the slides, including successively the steps of permeabilization, blocking, incubation with primary antibodies (PAX6 for staining neurons, TUBB3 for staining neural progenitors, S100B for staining astrocytes), incubation with secondary antibodies, counter-staining with DAPI, then the final mounting of the slides;

[0120] 4- The samples were observed under a fluorescence microscope (Figure 14B). Numerous neurons with axonal extensions are visible (TUBB3). Neural progenitors (PAX6) and astrocytes (S100B) are also present in significant numbers and exhibit satisfactory morphology.

[0121] This observation (Figure 14B) highlights the presence and viability of all cell types characteristic of the target organ, in this case the brain, after cell dissociation and seeding within a multi-compartment microfluidic device. The method according to the invention thus enables the formation of a biological model derived from the brain, to which a stimulus can be applied, and the effects of said stimulus can be evaluated and considered representative of effects observable in vivo.

[0122] The invention also relates to a compartmentalized microfluidic device 1 for implementing the process according to the invention.

[0123] The inventors have developed a multi-compartment microfluidic device 1 for culturing dissociated cells, obtained after cellular dissociation from the biological structure derived from the target organ, as well as for culturing one or more distinct neuronal cultures, enabling the proliferation of their nerve endings and the recording of the functional activity of neurons present within the device. In this sense, as illustrated in particular in Figures 3 to 5B, the device 1 according to the invention has a casing-like shape delimiting a volume within which at least two distinct cellular compartments coexist. More specifically, a first embodiment of the invention comprises three distinct compartments (10, 20, 40) (Figures 3 to 10). The second exemplified embodiment of the invention comprises only two distinct cellular compartments (10, 40) (Figures 11 and 12).The third illustrated embodiment comprises three distinct and aligned cell compartments (10, 40, 60) (Figure 13). The internal volume defined by this device 1 is delimited by the lid 2 and the base 3.

[0124] The first cell compartment 10, as shown in all the figures, corresponds to an open well into which dissociated cells 100, originating from the initial biological structure derived from the target organ, are deposited onto a cell culture substrate 11 configured to promote the growth or quiescence of these cells 100 intended to form the biological model derived from the target organ. These cells 100 can be cultured to form a monolayer 103 of cells 100 (Figures 8 and 10) or a multilayer of cells 100 (Figures 9 and 12).

[0125] Advantageously, the cell culture substrate 11 of this first cell compartment 10 exhibits mechanical properties that allow control of the surface condition, porosity, viscosity, and roughness of the substrate. Furthermore, it is non-cytotoxic, as it promotes the growth or quiescence of dissociated 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). The cell culture substrate 11 can also include fibroblasts and / or immune cells, enabling it to mimic, for example, the composition and function of the basal lamina found in target organs, such as various skin models.As illustrated in all the figures, this cell culture substrate 11 may have a smooth surface, thus facilitating the application of the solution containing the dissociated cells 100. Alternatively, according to a variant not shown, the cell culture substrate 11 may include a plurality of protrusions (micro- or macro-shaped) projecting from its surface. Alternatively, the surface of this cell culture substrate 11 may also be pitted. In order to customize the biological model to be obtained after culturing the dissociated cells 100 and on which the effects of a chosen stimulus are to be evaluated, a biological sample 101 (such as microbiota) from the target organ, taken from an individual, can be applied to the cells 100 forming said biological model. This biological sample 101 can be positioned directly onto the cells 100 (Figure 10).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. An inlet and an outlet will then be provided within the device, allowing for the introduction of the solution containing the dissociated cells from the initial biological structure derived from the target organ and / or the renewal of the culture medium necessary for cell growth and obtaining the biological model derived from said target organ.

[0126] 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 dissociated cells 100 and the characterization of their behavior in response to the application of a given stimulus. The neurons 400 are distributed on a substrate 41 comprising a plurality of electrodes 44, which allows the digitization of the behavior of the cells 100, forming the biological model mimicking the target organ and its function, in response to said stimulus.

[0127] 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.

[0128] Advantageously, according to a third embodiment illustrated in Figure 13, the device comprises a third cellular compartment 60 into which neurons 600 from a second neuronal culture are introduced and cultured. These neurons are cultured to extend their respective neuronal terminals 602 to the second cellular compartment 40, via microchannels 71 of a second means forming the biological interface provided for this purpose, thus enabling the innervation of the neurons 400 of the first neuronal culture, thereby mimicking the functioning between the central and peripheral nervous systems.In this variant, the axons of the neurons 400 in the first neuronal culture can also extend their respective neuronal terminals to the third cellular compartment 60, via the aforementioned microchannels 71 of the second medium forming the biological interface, thus innervating the neurons 600 in the second neuronal culture and forming neuronal connections 601 between the two neuronal cultures. In this variant, additional electrodes 45 are provided at this third cellular compartment 60 and are configured to measure the functional activity of the neurons 600 in the second neuronal culture.

[0129] Advantageously, the device 1 according to the invention can be provided with a fourth cell compartment 20. This fourth cell compartment 20, as shown in Figures 3 to 10, 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 fourth 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 fourth cellular compartment 20 is located below the first cellular compartment 10 and is separated from it by the porous membrane 30, thus allowing the dissociated 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.

[0130] The porous membrane 30, as shown in Figures 3 to 10, 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 fourth 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 3, 4 and 6 to 10), 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.

[0131] According to the variant shown in Figure 5A, the porous membrane 30 further comprises additional electrodes 45 present at the interface between the first cellular compartment 10 and the fourth cellular compartment 20. Such additional electrodes 45 allow, for example, the application of an electrical stimulus to the cells of the first cellular compartment 10 and / or the fourth cellular compartment 20 and the study of the cellular response to such a stimulus.

[0132] According to the variant of the invention shown in Figure 5B, 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 fourth cell compartment 20, so as not to impact the physiological exchanges between these two compartments.

[0133] Device 1 according to the invention works as follows.

[0134] The solution containing the dissociated cells 100 from the initial biological structure derived from the target organ whose function and environment are to be mimicked and reproduced is placed on the surface of the cell culture substrate 11 of the first cell compartment 10. These cells are cultured until they form the final biological model derived from said target organ, for example, by forming a monolayer 103 of cells 100 or a multilayer 104 of cells 100. When these cells have colonized the entire surface of the cell culture substrate 11 of the first cell compartment 10, it is possible to place a biological sample 101 of said target organ, taken from an individual, directly onto said dissociated cells 100.The biological sample 101 in question is advantageously from the microbiota of the patient's target organ, thus enabling the biological model reproduced within the first cellular compartment 10Tl to be functionalized.

[0135] while personalizing it to the individual from whom said biological sample 101 was taken. Alternatively, depending on the stimulus that one wishes to test, it is possible not to position any additional biological element on said cells 100.

[0136] In parallel, 400 neurons (for example, neurons consisting mainly of sensory neurons) are introduced and cultured within the second cellular compartment 40, with the aim of innervating the cells 100 forming the biological model reproduced in the first cellular compartment 10, via their respective neuronal terminals 402. To do this, the axons of the 400 neurons grow through the fluidic microchannels 51, thus forming the neuronal connections 401 allowing communication by neuronal connection between the first cellular compartment 10 and the second cellular compartment 40.In the case where the dissociated cells 100 include neurons, the axons of the latter can also develop through the fluidic microchannels 51 and thus participate in the formation of neuronal connections 401 allowing communication by neuronal connection between the first cellular compartment 10 and the second cellular compartment 40. As already mentioned, these neurons 400 are distributed on a cell culture substrate 41 comprising a plurality of electrodes 44 allowing the functional activity of said neurons 400 to be measured.

[0137] As previously stated, additional neurons 600, belonging to a second neuronal culture and advantageously comprising glutamatergic neurons 600, can be introduced and cultured within a third cell compartment 60, with the aim of innervating the neurons 400 of the first neuronal culture present in the second cell compartment 40. To achieve this, the axons of the neurons 600 from the second neuronal culture and / or the neurons 400 from the first neuronal culture grow through the fluidic microchannels 71, thus forming neuronal connections 601 enabling neuronal communication between the second cell compartment 40 and the third cell compartment 60. These neurons 600 are distributed across a third cell culture (not shown in Figure 13) comprising a plurality of electrodes 45 for measuring the functional activity of said neurons 600.

[0138] In the advantageous embodiment of the invention shown in Figures 3 to 10, in which the device 1 includes a fourth cell compartment 20, endothelial cells 200 are introduced and cultured within this fourth cell compartment 20 in order to colonize all of its walls, thus reproducing a blood vessel and enabling the device 1 according to the invention to ensure a vascularization function of the biological model derived from the target organ reproduced in the first cell compartment 10. Such a fourth cell compartment 20 can also be provided under the second cell compartment 40 and / or under the third cell compartment 60 so as to reproduce, respectively, the vascularization of the peripheral nervous system (PNS) and the central nervous system (CNS).

[0139] 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 cells 100 forming the biological model derived from the target organ and, more generally, of all the biological elements present in the first cellular compartment 10 forming the biological model derived from the target organ to be mimicked, to said stimulus by measuring the functional activity of all the neurons present in device 1. These neurons can be those present in the dissociated cells 100, the neurons 400 of the first neuronal culture and, if applicable, the neurons of the second neuronal culture. The functional activity of the neurons present is measured via electrodes 44, 45 distributed throughout the device and connected to a measuring instrument (not shown).

[0140] 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 dissociated cells 100 forming the biological model derived from the target organ that one wishes to reproduce. Such a stimulus provokes reactions within the first cellular compartment 10, particularly within the cells 100.These reactions result in modifications to the electrical activity of neurons present in this biological model, when it contains them, of the neuronal terminals 402 of the neurons in the first neuronal culture, and, if applicable, of the neuronal terminals 602 of the neurons 600 in the second neuronal culture. This activity is recorded by electrodes 44 and 45 and subsequently analyzed to obtain a derived model of neuronal functional activity. Alternatively or cumulatively, the application of this chemical or biological stimulus can be indirect, by applying it within the fourth cellular compartment 20, which can then be transmitted to the cells 100 of the first cellular compartment 10 via the intercalated porous membrane 30. Furthermore, the influence of vascularization (e.g., the bioavailability of a new molecule) on such a stimulus can be assessed.

[0141] 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 dissociated cells 100 forming the biological model derived from the target organ that one wishes to reproduce. It is thus possible to evaluate the reactions within the first cellular compartment 10, in particular the 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 an electric current, in order to evaluate the reactions within the first cellular compartment 10. [List of references]

[0142] 1: Multi-compartment microfluidic device

[0143] 2: Lid of the multi-compartment microfluidic device

[0144] 3: Base of the multi-compartment microfluidic device

[0145] 10: first cellular compartment

[0146] 11: Cell culture substrate

[0147] 20: fourth cellular compartment

[0148] 21: Entry of the fourth cellular compartment

[0149] 22: Exit from the fourth cell compartment

[0150] 30: porous membrane

[0151] 31: separate membrane incorporating the substrate of the second cellular compartment; 40: second cellular compartment

[0152] 41: Cell culture substrate of the second cell compartment covered with electrodes

[0153] 42: entrance to the second cellular compartment

[0154] 43: exit from the second cell compartment

[0155] 44: electrode(s)

[0156] 45: Additional electrode(s)

[0157] 50: first means forming the biological interface between the first cellular compartment and the second cellular compartment

[0158] 51: Fluidic microchannel

[0159] 60: third cellular compartment

[0160] 71: Fluidic microchannel of the second medium forming the biological interface between the second cellular compartment and the third cellular compartment

[0161] 100: cells dissociated from the initial biological structure

[0162] 101: biological sample (microbiota) of said target organ taken from an individual; 103: monolayer of cells forming the biological model

[0163] 104: multilayer of cells forming the biological model

[0164] 200: endothelial cell(s)

[0165] 400: neuron(s) forming the first neuronal culture; 401: neuronal connection(s) between cells of the first cellular compartment and the second cellular compartment

[0166] 402: neuronal terminal(s) of the neurons forming the first neuronal culture; 600: neuron(s) forming the second neuronal culture

[0167] 601: neuronal connection(s) between cells of the second cellular compartment and the third cellular compartment

[0168] 602: neuronal terminal(s) of the neurons forming the second neuronal culture

Claims

32 DEMANDS 1. An in vitro method for evaluating the effects of a stimulus on a biological model derived from a target organ, said method comprising the following steps: a) Cellular dissociation of a biological structure derived from said target organ, said biological structure comprising cells (100) forming a 3D structure (A), b) Culturing of the dissociated cells (B), c) Obtaining the biological model derived from said target organ (C), d) Application of said stimulus to the biological model derived from said target organ (D), e) Recording of the functional activity of neurons present within said biological model and / or neurons (400) of a first neuronal culture and / or neurons (600) of a second neuronal culture, the nerve endings (402) of the neurons present within said biological model and of the first neuronal culture forming a neuronal connection (401) between the cells (100) of said biological model and the neurons (400) of the first neuronal culture, the nerve endings (602) of the neurons (400) of the first neuronal culture and of the neurons (600) of the second neuronal culture forming a neuronal connection (601) between the neurons (400) of the first neuronal culture and the neurons (600) of the second neuronal culture, obtaining a derived model of neuronal functional activity (E).

2. Method according to claim 1, comprising a step (F), prior to step (D), of recording the functional activity of neurons present within said biological model and / or neurons (400) of a first neuronal culture and / or neurons (600) of a second neuronal culture, and of obtaining a reference model of neuronal functional activity.

3. Method according to claim 2, comprising a step (G) of comparison between the reference model of neuronal functional activity and the derived model of neuronal functional activity.

4. A method according to any one of the preceding claims, wherein step (A) comprises the use of a chemical compound and / or a biological compound to destroy the intercellular bonds of the biological structure while preserving the integrity of the cells and their function.33 5. A method according to any one of the preceding claims, wherein step (B) further comprises culturing said first neuronal culture so that the nerve endings (402) of the neurons (400) of this first neuronal culture innervate the cells (100) of the biological model.

6. A method according to the preceding claim, wherein step (B) further comprises culturing said second neuronal culture so that the nerve endings (602) of the neurons (600) of this second neuronal culture innervate the neurons (400) of the first neuronal culture.

7. A method according to any one of the preceding claims, wherein the cells (100) of the biological model form a monolayer (103) or a multilayer (104), said cells (100) preferably being derived from induced pluripotent stem cells, preferably from human induced pluripotent stem cells.

8. Multi-compartment microfluidic device (1) for implementing the method according to any one of the preceding claims, said device (1) comprising: a first cell compartment (10) comprising the biological model derived from said target organ, a first cell culture substrate (11) forming a surface on which said biological model is disposed and being configured to promote the growth or quiescence of the cells (100) of said biological model, a second cellular compartment (40) comprising the first neuronal culture, a second cell culture substrate (41) forming a surface on which said first neuronal culture is disposed and being configured to promote the growth or quiescence of the neurons (400) of said first neuronal culture, and at least one first means (50) forming a biological interface to allow communication by neuronal connection between the first cellular compartment (10) and the second cellular compartment (40).

9. Device (1) according to the preceding claim, further comprising: a third cellular compartment (60) comprising the second neuronal culture, a third cell culture substrate forming a surface on which said second neuronal culture is disposed and being configured to promote the growth or quiescence of the neurons (600) of said second neuronal culture, and at least one second means forming a biological interface to allow communication by neuronal connection between the second cellular compartment (40) and the third cellular compartment (60).

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