EP3728556A1

EP3728556A1 - Microfluidic chip, microfluidic lab-on-a-chip, method for producing such a chip and analysis method

Info

Publication number: EP3728556A1
Application number: EP18845401.1A
Authority: EP
Inventors: Sarah Milgram; Damien FLEURY
Original assignee: Nanobiose SAS
Current assignee: Nanobiose SAS
Priority date: 2017-12-21
Filing date: 2018-12-21
Publication date: 2020-10-28
Also published as: WO2019122788A1; US20200406263A1

Abstract

The microfluidic chip (1) comprises: • at least one inlet channel (4) connected to at least one well (2), each well (2) being associated with an outlet channel (5), • at least one analysis chamber (3a, 3b) connected to at least one well (2). • an analysis surface (6) comprising collection elements (6a) collecting biomarkers present in the liquid originating from the well (2) and representative of the cellular response of a biological sample contained in the well (2). A flow of liquid flows from the well (2) towards the analyse surface (6) comprising the collection elements (6a). The flow of liquid flows at between 0.1µL/min. and 2 ml/min. and flows in a laminar manner through the analysis chamber (3a, 3b). The chip (1) is part of a microfluidic lab-on-a-chip provided with a flow generator applying a plurality of different liquids.

Description

Micro-fluidic chip, on-chip-micro-fluidic laboratory, method for manufacturing such a chip and analysis method

Technical field of the invention

The invention relates to a microfluidic chip, to a microfluidic on-chip laboratory, to a process for manufacturing a microfluidic chip and to an analysis method.

State of the art

To detect, identify and monitor the response of cells to biological stimuli (viruses, bacteria, etc.), chemical (drugs, etc.) or mechanical stimuli (application of a flow through cells, pressure, shear stress, etc.), a cytokine profile can be established. For that, one must know the nature of the cytokines secreted by the cells, their quantity as well as the kinetics of cytokine secretion.

At the present time, the techniques and equipment available to carry out this type of study are complex and require many experimental steps which makes these approaches of little use from an industrial or quasi-industrial point of view. The cells must first be cultured before the result of a reaction is taken and analyzed. The analysis will then include several stages of marking, washing, revelation and analysis. These steps are both time consuming and expensive. The result is only accessible for several hours, or even several days after the completion of the cell biology experiment.

Also, to reduce the duration of the analyzes and their cost, the quantity of samples analyzed and their exposure time are reduced to the strict minimum. However, when the quantity of samples and / or the analysis time are too small, this can lead to results that do not reflect the real response of the immune system because the equipment and / or protocols are not suitable. To remedy this problem, different devices are developed. According to the approach proposed in the document entitled "Reconfigurable microfluidic device with integrated antibody arrays for capture, multiplexed stimulation and cytokine profiling of human monocytes", Vu et al., Biomicrofluidics 9, 04415 (2015), several cell populations are captured and organized in two dimensions on specific areas of an analysis surface. An infusion system mounted on the assay surface allows for multiplexing, i.e., it is chosen to simultaneously observe the cytokine response generated by several types of biochemical stimuli after the infusion. Cytokines produced in response to different stimuli are captured on other specific areas of the assay surface and revealed by specific reagents. The capture of cells by their membrane receptors and their organization in 2D creates a cell culture environment substantially removed from physiological conditions.

According to another axis of improvement, Prot et al. propose in the publication "Integrated proteomic and transcriptomic investigation of the acetaminophen toxicity in liver microfluidic biochip" (PLoS One, 2011. 6 (8): p.212-68) to use microfluidics in order to cultivate liver in a three-dimensional environment and under continuous infusion of nutrients and toxic molecules. The analyzes carried out a posteriori make it possible to quantify the toxicity of the tested molecules more accurately and more surely than under the conditions of culture in 2D and without infusion. The results obtained in 3D were indeed closer to those observed in laboratory animals.

Furthermore, in the publication "High-Content Quantification of Single Cell Immune Dynamics" (Cell Reports, Volume 15, 2016), Junkin et al. disclose a microfluidic device in which several biological tests can be performed in parallel, and wherein the cells are placed under perfusion so as to perform dynamic analyzes. The microfluidic system is coupled to a fluorescence microscope to observe in real time the response of several individualized cells, that is to say isolated from each other. These microfluidic devices attempt to improve the conditions for analyzing the immune response of a cell culture. But these improvements are not sufficient to be in line with the expectations of the market, which wants to obtain a low-cost device, allowing the realization of complex analyzes, and providing fast and reliable results.

Object of the invention

An object of the invention is to provide a micro-fluidic chip for knowing the response of a cell sample by carrying out complex analyzes associating a more physiological cellular environment and a multiparametric detection of biomarkers continuously and providing a more usable result. quickly than with the solutions proposed by the prior art.

The chip is remarkable in that it comprises:

A first well intended to receive a biological sample, the substrate defining sidewalls and at least one bottom or top wall of the first well, the first well opening on the surface of a face of the substrate,

A first inlet channel intended to supply the first well with a first liquid, the first inlet channel ending with a first inlet opening into the first well,

A first outlet channel having a first end formed by a first outlet of the first well for discharging the first liquid,

A first analysis chamber having a first analysis input connected to the first well by means of the first output channel and an evacuation outlet opening into the first analysis chamber and distinct from the first analysis input; first analysis chamber having a bottom formed by the substrate,

in that the first inlet and the first outlet of the first well define a different first axis and not parallel to a second axis defined by the first analysis input and the discharge outlet of the first analysis chamber, and in that the first analysis chamber is closed by an analysis wall comprising capture elements configured to capture at least one biomarker of the first liquid flowing in the first analysis chamber, the analysis wall being mounted in detachable manner with respect to the substrate.

Advantageously, the first well is closed by means of a cover layer distinct from the analysis wall so as to be able to dismount the analysis wall independently of the covering layer.

In one development, the cover layer is removably mounted relative to the face of the substrate.

In a particular embodiment, the cover layer defines at least one through hole forming a ring surrounding the outlet of the first outlet channel, the ring defined in the cover layer forming completely or partially the side walls of the first chamber. analysis, the substrate face forming a bottom wall of the first analysis chamber.

In a preferred configuration, the side walls of the first analysis chamber are partially formed in the substrate, the first analysis chamber being formed by a first cavity emerging on the face of the substrate, the covering layer defining a ring surrounding the first cavity and partially forming the side walls of the first analysis chamber.

In an alternative embodiment, the sidewalls of the first output channel are formed by a groove in the face of the substrate, the cover layer enclosing the first well and the first output channel to the first analysis chamber.

In an advantageous configuration, the first outlet channel is defined by a groove formed in the cover layer which closes the first well. Preferably, an additional channel is connected to the first output channel between the output of the first well and the first analysis chamber. The additional channel is advantageously formed by an additional groove in the face of the substrate and / or in the cover layer.

It is advantageous to provide that the first analysis chamber comprises first and second analysis inputs respectively defining first and second liquid introduction directions in the first analysis chamber. The first and second analysis inputs are arranged so that the first and second liquid introduction directions intersect in the first analysis chamber.

Preferably, the chip comprises an evacuation connected to the evacuation outlet of the first analysis chamber and the first and second septums configured to seal the first inlet channel and the evacuation.

In another development, the distance separating the analysis wall and the bottom of the first analysis chamber is less than 2 mm.

The invention also relates to a lab on a chip which is compact while remaining particularly effective.

The micro-fluidic on-chip laboratory is remarkable in that it comprises:

A micro-fluidic chip according to one of the preceding embodiments, the microfluidic chip comprising a covering layer enclosing the first well and an analysis wall closing the first analysis chamber in a removable manner, the wall of analysis being provided with capturing elements configured to capture at least one biomarker of the liquid flowing in the first analysis chamber and from the first well;

A flow generator connected to the first input channel of the microfluidic chip, the flow generator being configured to emit at least a first liquid flow intended to pass through at least the first well filled with a cell sample and the analysis chamber, the first flow of liquid having a flow rate of between 0.1 mI / h and 2 mL / min so that the flow of liquid flowing in the first analysis chamber is laminar in at least half of the first analysis chamber having the exhaust outlet.

In a development, the flow generator is connected to an additional channel of the microfluidic chip and wherein the flow generator is configured to:

Applying a first flow on the first inlet channel so as to feed the first well during a first period, the first flow comprising a first liquid comprising at least nutrients,

Applying an additional stream on the additional channel, the additional stream comprising reagents configured to react with biomarkers characteristic of a cellular response of the biological sample contained in the first well, the capture elements being configured to capture and / or react with biomarkers.

The invention also relates to a method for producing a chip that is easy to implement.

The process is remarkable in that it comprises the following successive steps:

supplying a microfluidic chip according to one of the preceding embodiments,

- Close the first well by means of a cover layer which at least partially forms the side walls of the first analysis chamber,

- Close the first analysis chamber by means of an analysis wall comprising capturing elements configured to capture constituents of the liquid flowing in the first analysis chamber.

Another object of the invention is to provide a method of analyzing a cell culture by means of a microfluidic chip. The method makes it possible to carry out complex analyzes, quickly and efficiently, in order to know the response of a cell culture to several types of stimuli (chemical substance, biochemical, biological, pharmaceutical, mechanical stress, radiation, material, etc.).

For this purpose, the method for analyzing a cell culture comprises:

Provide a microfluidic chip comprising:

a first well filled with a cell sample emitting a first cellular response containing at least one biomarker;

a first analysis chamber having a first analysis input fluidly connected to the first well by means of a first output channel and an evacuation outlet opening into the first analysis chamber and distinct from the first input of analysis,

o capturing elements disposed on a wall of the first analysis chamber, the capturing elements being configured to capture at least one representative biomarker of the first cellular response flowing from the first well to the first analysis chamber;

Applying a first stream of liquid supplying the first well so that the first cellular response is taken from the first well to the evacuation outlet through the first analysis chamber, said at least one biomarker flowing in the first chamber; analysis chamber to be captured by the capturing elements,

• analyze the capturing elements.

According to a particular embodiment of the invention, the method comprises the provision of at least one revelation reagent configured to react with said at least one biomarker representative of the first cellular response captured by the capture elements, said at least one revealing reagent passing through the first analysis chamber simultaneously or after the passage of said at least one biomarker in the first analysis chamber, the analysis of the capture elements being carried out by searching for said at least one revelation reagent. Preferably, the method comprises applying a second stream to an additional input of the microfluidic chip, the additional input opening into the first output channel through a first junction located between the first well and the first analysis chamber. so that the second stream enters the first analysis chamber, the second stream comprising said at least one developing reagent so that said at least one developing reagent is devoid of contact with the cell sample.

According to a specific embodiment, the method comprises filling the first output channel, after the first junction, alternatively by the first stream comprising the said at least one biomarker and the second stream comprising the said at least one revelation reagent.

According to an alternative embodiment, the first junction is associated with a mixer configured to mix the first stream with the second stream in the first output channel after the first junction.

In another development, the microfluidic chip comprises a second well comprising an additional cell sample emitting a second cellular response with at least one additional biomarker, the second well being fluidly connected to the first analysis chamber by a second output channel. joining the first output channel before the first analysis chamber, the flow generator supplying the first and second wells so as to take the representative biomarker and additional biomarker respectively of the first and second cellular responses in the first analysis chamber and delivering first and second laminar flows within the first analysis chamber, the biomarker and the additional biomarker being respectively present in the first and second laminar flows.

In a particular embodiment, the first well is filled with the cell sample before closing the first well by a cover layer, the cover layer at least partially forming the side walls of the first analysis chamber.

Advantageously, the first analysis chamber is closed by means of an analysis wall comprising the capturing elements.

Preferably, the analysis method comprises disassembly of the analysis wall to perform the analysis of the capturing elements out of the first analysis chamber.

In a particular embodiment, the chip is defined in a substrate A, the substrate A defining a bottom and a side wall of the first well and a groove forming the first outlet channel and in which the analysis method comprises the closure of sealing manner of the first well and the first outlet channel by means of a removable cover layer.

According to an alternative embodiment, the cover layer defines an opening forming a closed ring around the first analysis chamber and wherein the method comprises, after the installation of the cover layer, the installation of a surface analyzer closing the first analysis chamber in a sealed manner, the analysis surface comprising the capturing elements.

According to a specific embodiment, the side walls of the first outlet channel are at least partially formed in the cover layer.

Brief description of the drawings

Other advantages and features will emerge more clearly from the following description of particular embodiments of the invention given by way of non-limiting example and represented in the accompanying drawings, in which:

FIG. 1 represents a microfluidic chip according to a first embodiment, FIG. 2 represents a microfluidic chip according to a second embodiment,

FIG. 3 represents an exploded view of a microfluidic chip according to one embodiment of the invention,

FIG. 4 represents a microfluidic on-chip laboratory using the chip of FIG. 3 after assembly of the various parts, as well as a flow generator, the flow being represented by arrows at the inputs.

FIG. 5 represents another embodiment of a microfluidic chip,

FIGS. 6 and 7 show other embodiments of a microfluidic chip.

detailed description

A microfluidic chip can advantageously be used in the context of biomedical and / or biological analyzes, for example to understand the impact of chemical, biological and / or mechanical stresses on the immune system. It is also possible to track the impact of a material or radiation.

As illustrated in the various figures, the microfluidic chip 1 comprises at least a first well 2a intended to receive a biological sample composed in part of living cells, for example: tissues, a biopsy, organoids and / or a cell culture eukaryotic or prokaryotic. For example, it is considered that the chip 1 is associated with a cell culture in the following description. The first well 2a has a bottom, a side wall and a top. The first well 2a is a cell culture well, that is to say that it is intended to contain cells to be analyzed or that it is intended to contain cells of a cell culture.

The first well 2a has a first inlet which opens into the first well 2a and a first outlet which opens into the first well 2a and distinct from the first inlet. Thus, it is possible to have a flow of liquid that passes through the first well 2a to approach the in-vivo conditions. The first well 2a is made in a substrate A which defines the side walls of the well and preferably the bottom and / or the top of the well 2a. According to the embodiments, the substrate A can be formed by a single material or by several materials, for example by a stack of several different layers. Substrate A is preferably monoblock.

The micro-fluidic chip 1 further comprises a first analysis chamber 3a having a first analysis input. The first analysis chamber 3a is intended for the analysis of molecules present in the liquid from the well 2a and representative of the cellular response.

The microfluidic chip 1 comprises a first input channel 4a intended to feed the first well 2a. The first input channel 4a opens into the first well 2a by means of the first input. The first input channel 4a is intended to be connected to a flow generator which feeds the microfluidic chip 1 with a liquid or several different liquids. The first input channel 4a ends with the first input.

The chip further comprises a first outlet channel 5a for discharging the first liquid out of the well 2a. The first output channel 5a has a first end formed by the first output of the well 2a. The inlet channel 4a, the first well 2a and the first outlet channel 5a are connected in series according to the flow of the fluid.

The first analysis chamber 3a is connected to the first well 2a by means of the first output channel 5a. The first analysis chamber 3a has a first analysis input disposed at a first end of the chamber 3a and a first evacuation outlet which is disposed remote from the first analysis input. The first analysis input and the first evacuation output define the flow of the liquid within the first analysis chamber 3a. Advantageously, the first discharge outlet is disposed at an end opposite to the first analysis inlet. The first analysis chamber 3a is dissociated from the first well 2a to facilitate the analysis and possibly not to disturb the cells present in the first well 2a with reagents used to observe and / or analyze the cellular response. The analysis chamber 3a receives the liquid from the well 2a and comprising elements representative of the cellular response or biomarkers by means of the first output channel 5a so that the analysis chamber 3a receives continuously information representative of the response. cellular. Preferably, the analysis chamber 3a is separated from the first well 2a by an outlet channel which represents a volume of liquid less than 100mί so that the information produced by the first well 2a arrives quickly in the analysis chamber 3a. This configuration makes it possible to follow, in time, the reaction of the cells in the well 2a by limiting the disturbance introduced by the analysis of the cellular response.

The analysis chamber 3a is closed by an analysis wall 6. The analysis wall 6 is in contact with the liquid which passes through the analysis chamber and which comes from the well 2a. The analysis wall 6 has an analysis surface on which capturing devices or probes 6a are installed which capture certain elements of the liquid which circulate in the analysis chamber 3a in order to allow their detection.

The well 2a has reduced dimensions in order to reduce the quantity of cells used as well as the quantity of reagents to be used. However, to obtain or measure a signal sufficient to perform the analyzes, it is necessary to provide a well configuration 2a which facilitates the stimulation of the cells and / or the recovery of the products resulting from the reaction of the cells without the need to apply significant pressure. in the chip and in particular in the well 2a. Advantageously, the volume of the first well 2a is less than 1 ml, which makes it possible to limit the size of the well without penalizing the measurement. Also advantageously, the volume of the first well 2a is greater than 5mI. It is particularly advantageous to use a well allowing the installation of a three-dimensional cell culture support in order to approach in vivo conditions. In addition to the three-dimensional organization of the cells which is more physiological, the advantage of the culture supports or three-dimensional matrices is to increase the culture surface per unit volume of the well. This makes it possible to have more cells secreting biomarkers and therefore more biomarkers per unit volume, and thus to reduce the limit of detection of the cellular response. It is particularly advantageous to use a textured or porous support to increase the culture surface. Each of the three dimensions of the well 2a is greater than 100mΐti and preferably less than 10mm. The height of the well is advantageously greater than or equal to the hydraulic radius of the well.

The inventors propose judiciously placing the liquid inlet and the liquid outlet to ensure a homogeneous flow in the majority of the well in order to have a cellular response close to the in-vivo conditions.

The first input channel defines a first flow direction of the liquid. The first output channel defines a second flow direction. The inventors have observed that to have a greater efficiency in the stimulation of the cell culture, it is advantageous to provide that the first and second flow directions are not aligned. Thus, the nutrients and the test molecules are better distributed within the first well 2a.

When the input and output channels have an axis of revolution, the directions of flow are defined by the axes of revolution.

The offset of the flow directions makes it possible to limit the zones of stagnation of liquid in the well 2, that is to say the dead volumes. The proportion of the biological sample operating in a static state is reduced, which improves the quality of the cellular signal obtained. This configuration makes it possible to ensure that the products coming from the input are brought into contact with the majority of the cells. Advantageously, the inlet and the outlet of the first well 2a define a different and non-parallel first axis of a second axis defined by the inlet and the outlet of the analysis chamber 3a. The inlet and outlet of the well 2a are advantageously arranged so that the flow inside the well 2a moves mainly along a longitudinal axis of the well, for example, its axis of revolution. In this configuration, the cells disposed in the well 2a are in contact with the liquid flow, which notably allows a better cellular response and therefore a more reliable analysis with a small amount of cells used.

This configuration makes it possible to couple a three-dimensional cell culture well 2 with a two-dimensional analysis chamber 3a. The analysis chamber 3a has a ratio between the chamber volume and the surface of the side walls of the chamber 3a which is significantly larger than the ratio between the well volume and the surface of the side walls of the well 2a. This configuration maximizes the area of the top and bottom walls of the analysis chamber per unit volume. This makes it possible to optimize the use of an analysis surface by promoting the contact of the molecules to be detected with the capture devices 6a present on the analysis surface. The main advantage of a flat or substantially flat analysis surface is the compatibility with conventional optical detection instruments. In addition, such a surface analysis facilitates the possibility of multiplexing the detection, that is to say the possibility of multiplying the number of different molecules detected by multiplying the number of different capture devices 6a present on the surface of the detector. analysis.

The first inlet and the first outlet may open into a bottom, top or side wall of the first well 2a near the bottom or top.

The bottom of the well 2a is separated from the top of the well 2a by a first distance along the longitudinal axis. It is advantageous to provide that the first input is separated from the first output by a distance at least equal to half of the first distance along the longitudinal axis. To facilitate cell stimulation and recovering the cellular response, even more advantageously, the input is separated from the output by a distance of at least 75% of the first distance along the longitudinal axis.

Preferably, the first outlet comes into contact with a bottom wall or top of the well 2a when the latter is closed. As an alternative, the first outlet is formed in a top or bottom wall and preferentially along an axis that is not perpendicular to the longitudinal axis.

In a particular embodiment, the first outlet and the first inlet are disposed on side walls of the well 2a. This embodiment is particularly advantageous for observing the cell culture by microscopy.

In an advantageous embodiment, the liquid entry is carried out in the bottom of the well 2a. It is advantageous to have an inlet channel 4a whose longitudinal axis is non-parallel, for example perpendicular to the longitudinal axis of the outlet channel 5a. However, this embodiment makes it more difficult to observe the cell culture optically.

In an advantageous embodiment, in a cutting plane perpendicular to the height of the well 2a, the well has transverse dimensions between 100miti and 10mm to limit the volume of the well 2a and the size of the well 2a. The volume of the well 2a being reduced, the same is true of the quantity of cells.

Advantageously, the well 2a is a cavity having a volume of revolution. The section of the cavity may be square, rectangular, circular, ovoid or any. The section is observed in a first plane which is advantageously parallel to the upper and / or lower surface of the substrate which defines the well. Advantageously, the well 2 is a right circular cylinder which provides a configuration closer to in-vivo conditions. In a particular embodiment, the walls of the well 2a or at least a portion of the walls of the well 2a are covered by a coating configured to allow the cells to adhere and maintain them inside the well 2a. According to the configurations, only the side walls are covered with a coating configured to allow adhesion of the cells or alternatively the side walls and the top are covered by the coating. It is still possible to provide that the bottom is also coated with coating for the adhesion of the cells.

Alternatively or in addition, the cells are cultured in one or more three-dimensional supports placed inside the well 2a so as to create a three-dimensional cell culture structure. The three-dimensional supports may be matrices or gels depending on the type of cells studied. The fact of realizing three-dimensional cell cultures makes it possible to approach the conditions in vivo, and thus to get closer to the real response of a living organism.

The first analysis chamber 3a has a bottom, side walls and a top. The first analysis chamber 3a collects the liquid coming out of the well 2a and the constituents of this liquid are captured on the analysis surface 6a. The constituent (s) are representative of the cellular response, that is to say of the response of the cell culture.

Preferably, the analysis chamber 3a is configured to have a larger width than that of the outlet channel 5a which comes from the well 2a.

The increase of the width is measured in a direction perpendicular to the transit axis of the liquid flow in the analysis chamber 3a and preferably in a horizontal direction in operation.

In a particular embodiment, the depth of the analysis chamber 3a is identical to the depth of the outlet channel 5a. The depth is measured perpendicular to the bottom of the analysis chamber and / or the analysis surface. In an alternative embodiment, the depth of the analysis chamber 3a is greater than the depth of the outlet channel 5a. In another embodiment, the depth of the analysis chamber 3a is less than the depth of the outlet channel 5a.

In a particular embodiment, the bottom of the first analysis chamber 3a is coplanar with the bottom of the first outlet channel 5a, which makes it easier to obtain a laminar flow in the analysis chamber. Alternatively, the bottom of the chamber 3a presents an obstacle, for example a step or ramp which facilitates the bringing of liquid into contact with the analysis surface. The step or ramp is defined so that the flow of liquid encounters an obstacle that forces the flow of liquid to approach the opposite wall of the analysis chamber 3a, that is to say the surface of analysis which contains the capturing devices 6a.

When the analysis chamber 3a comprises first and second analysis inputs or at least two analysis inputs as illustrated in FIG. 5, it is particularly advantageous to provide for two inputs to be configured to introduce two streams of liquid. in the analysis chamber 3a in two secant introduction directions. In this way, the two streams of liquid intersect inside the chamber 3a. Advantageously, the two inputs are fed by the same channel and provide the same liquid which preferably comprises biomarkers representative of the cellular response and / or a revelation reagent.

Alternatively, the two liquids are different. For example, it is possible to provide a liquid flow comprising biomarkers and a liquid flow comprising a revelation reagent.

This crossing of the streams has the effect of increasing the thickness and the width of the liquid flow in the analysis chamber 3a and thus facilitating a contact between the liquid which contains the cellular response and the analysis surface which is configured to capture representative elements of the cellular response. It is particularly It is advantageous to use this configuration when the thickness of the analysis chamber 3a is greater than the thickness of the outlet channel 5a and / or when the width of the analysis chamber 3a is greater than the width of the outlet channel. 5a.

In a particular embodiment illustrated in Figures 1, 2 and 5, the first well 2a and the first analysis chamber 3a are formed in a substrate A. The first analysis chamber 3a is formed by a cavity opening on one side of the substrate A.

In a particular embodiment, the well 2a does not have a bottom or apex formed by the substrate A so as to form a blind cavity in the substrate. In this configuration, it is easier to insert a three-dimensional support receiving the cell culture. The well 2 is closed by means of a cover wall 8, advantageously in a sealed manner. In other words, the first well 2a is formed in a substrate A and has a removable top formed by a cover layer 8 covering one side of the substrate A. The well 2a can be filled easily and then closed by the cover layer 8.

In the embodiment illustrated in the various figures, if the substrate A comprises several wells, the cover layer 8 is common to all the wells 2 as this facilitates the realization of the chip 1. However, it is also possible to provide that each well 2 is closed by a specific cover layer 8 in order to adapt to the cell culture or that a same cover layer 8 can be used for several wells 2. According to the embodiments, a cover layer 8 closes a or more than one well 2.

Alternatively, the cover layer 8 is used to close the well or wells, but it is not removed to fill the wells. It is then possible to inject the cell culture through the cover layer 8, for example by means of a syringe. Such a configuration makes it easier to manufacture the chip and especially its use. In yet another embodiment, the well 2 has a bottom and a side wall formed in a substrate A. The well is closed by a cover layer 8 distinct from the substrate A and not removable. The cells and their culture support are then placed in the well 2 by means of the inlet 4 and / or outlet 5 channels or by injection through the cover layer 8 of the well 2, for example to the using a syringe.

Advantageously, the microfluidic chip 1 is formed monolithically in a substrate which defines the first well 2a, the inlet channel 4a, the outlet channel 5a and optionally the first analysis chamber 3a. It is also advantageous to form the channel connecting the outlet of the analysis chamber 3a with a discharge 7 inside the substrate A.

In order to guarantee the reliability of the measurements, the micro-fluidic chip 1 is made of a biocompatible material which allows an optimal viability and cellular functionality approaching in vivo conditions. The material of the micro-fluidic chip 1 may be chosen from polymethyl methacrylate, polydimethylsiloxane, polyetheretherketone, cycloolefin copolymer, polycarbonate, polystyrene, a mixture containing Exo-1, 7.7 trimethylbicyclo [2.2.1] hept-2-yl acrylate and tricyclodecane dimethanol diacrylate, for example MED610 ™, stainless steel, glass, etc. It can also be made in a 3D printing material having the property of being biocompatible, such as the MED610 ™.

In an unillustrated configuration, the output channel 5a is completely formed in the substrate. Alternatively, in the illustrated configuration, the outlet channel 5a is not closed in its upper part and the substrate A advantageously defines a groove which connects the top portion of the well 2 with the analysis chamber 3a. The cover layer 8 closes the first outlet channel 5a.

Advantageously, the cover layer 8 is covered with an adhesive material so as to be easily fixed against the face of the substrate A. According to the embodiments, the adhesive material may or may not cover the well 2a. If the adhesive material covers the well 2a, the adhesive material is biocompatible because it comes into contact with the cell culture.

In an advantageous embodiment, the cover layer 8 closes the well 2a and the outlet channel 5a. The cover layer 8 defines an access opening to the analysis chamber 3. Advantageously, the cover layer 8 forms a closed ring around the side wall of the first analysis chamber 3a. The cover layer 8 continuously covers the well 2a, the outlet channel 5a and the tower of the analysis chamber 3 which facilitates the installation of the analysis wall 6 which closes the analysis chamber 3a so that waterproof. The analysis wall 6 forms the top of the analysis chamber 3a which presents the analysis surface with the capturing devices 6a. In a particular configuration not illustrated, the cover layer 8 defines a non-through groove which forms the bottom of the first analysis chamber 3a.

In a preferred embodiment illustrated in FIGS. 6 and 7, the substrate A comprises a hole which is preferably blind to define the well 2a. In contrast, the substrate A does not define or not completely the side walls of the first analysis chamber 3a. The latter are formed by means of the covering layer 8. In the illustrated embodiment, the bottom of the well 2a, the side walls of the well 2a, the bottom and side walls of the channel 5a as well as the bottom of the chamber of 3a analysis are formed in the substrate A.

In the embodiment of FIG. 7, the thickness of the analysis chamber 3a is defined by the thickness of the covering layer 8.

Advantageously and illustrated in Figures 6 and 7, the outlet channel 5a flares when entering the analysis chamber 3a. The outlet channel 5a has side walls which allow an expansion of the flow before entering the analysis chamber 3a. Preferably, the widening of the outlet channel 5a is made so that it reaches the width of the analysis chamber 3a defined by the side walls of the cover layer 8. The end of the outlet channel 5a is formed by the end of the groove in the substrate A so that the liquid reaches the surface of the substrate A. The step or the ramp formed by the end of the groove makes it possible to force the liquid to be wet. the surface of analysis and thus to facilitate the measurement. It is particularly advantageous to provide that the analysis surface 6 comes opposite the outlet channel 5a to facilitate the introduction of a flow of laminar liquid in the analysis chamber 3a.

The bottom of the analysis chamber 3a is then disposed on at least two different planes, one of which corresponds to the upper face of the substrate A.

As illustrated in FIGS. 6 and 7, the analysis chamber 3a is formed by depositing the covering layer 8 on the substrate A. It is therefore possible to simply modulate the characteristics of the analysis chamber 3a by modulating the dimensions of the opening formed in the cover layer 8 and / or by modulating the thickness of the cover layer 8. This configuration is particularly advantageous because it limits the dead volume in the analysis chamber 3a.

In a particular embodiment not illustrated, the cover layer 8 is disposed on the surface of the substrate A to close the well 2a and the outlet channel 5a. The cover layer 8 is in one piece and has a blind cavity that defines the analysis chamber 3a.

The analysis wall 6 defines the analysis surface with its capturing elements or probes 6a configured to capture constituents of the liquid flowing in the first analysis chamber 3a. The analysis wall 6 is removably mounted. The analysis surface which contains the probes 6a is separated from the substrate A by the cover layer 8 to analyze the cellular response. Installation and removal of the analysis wall 6 can then be performed without disturbing the well 2a, that is to say by avoiding removing the cover layer 8 which closes the well 2a. The analysis surface is covered by the capturing elements. The capture elements 6a are configured to fix one or more biomarkers, that is to say the components of the fluid from the well 2a and representative of the cellular response that it is desired to analyze. The capturing elements 6a can also be configured to fix the revealing reagent and thus allow a positive revealing control. The analysis surface may include areas with positive revealing controls and / or areas with biomarker-specific sensors, as well as positional markers that are visible when acquiring the signal or measurement.

The biomarker is a measurable biological characteristic related to a process. The biomarker may be a molecule produced secreted or modified by the cells of the cell sample. The biomarker may be a chemical molecule, a fragment of DNA, RNA, a metabolite, a sugar, a lipid, a protein or a protein fragment that is specific for the cellular response. The probes 6a may comprise, for example, antibodies, proteins, lipids, aptamers, sugars, peptides, immunoglobulin fragments or oligonucleotides. In the analysis chamber 3, the constituents of the cellular response detected or biomarkers are preferably cytokines. Detecting cytokines continuously allows to establish a kinetic profile. It is advantageous to install several probes 6a which react with the same biomarker (s) to improve the quality of the analysis.

By using an analysis wall 6 removable from the rest of the analysis chamber 3, it is possible to adapt the detection to the cellular reaction during the experiment. The use of a removable wall 6 is particularly advantageous when the analysis surface comprises several different detection probes which are configured to distinguish different elements of the cellular response, that is to say react to different biomarkers or cell secretion. The removable analysis wall 6 allows an opening of the chamber to change the analysis surface 6 and possibly the probes 6a. For example, if the cellular response changes over time, it is possible to change the probes of the analysis surface to adapt the probes to the response, for example depending on the nature of the cytokines or their concentration so that the measurement can follow the dynamics of the cellular response. This prevents the saturation of the signal.

It is also possible that the probes 6a degrade over time. In order to monitor the cellular response over a relatively long period, it is advantageous to replace the analysis wall 6 comprising worn probes with a new analysis wall 6 comprising new probes.

By using a removable analysis wall, it is possible to install and dismount the analysis surface without disturbing the cells present in the well 2a.

In an alternative embodiment, the analysis wall 6a is attached to the cover layer 8. The analysis wall 6a and the cover layer 8 may be formed from the same or different materials.

If the chip comprises several analysis chambers, it is advantageous for the cover layer 8 to form a continuous ring around each analysis chamber 3. Preferably, the cover layer 8 comes into direct contact with the substrate A. The layer cover 8 forms a sealing element ensuring continuity of recovery between the well, the outlet channel and the tower of the analysis chamber. The cover layer 8 also forms a seal accommodating the surface of the substrate and the surface of the analysis wall 6.

In one embodiment, the cover layer 8 defines an opening that corresponds exactly to the shape of each opening that exists in the substrate and that defines an analysis chamber 3a so as not to disturb the flow of liquid that has been moving since the inlet of the analysis chamber 3a to the outlet of the analysis chamber 3a. The height of the analysis chamber 3a is defined at least in part by the thickness of the covering layer 8. In order to ensure homogeneous diffusion of the flow in the analysis chamber 3a, the height of the side wall of the chamber 3a analysis may be less than half the difference between the width of the analysis chamber 3a and the width of the outlet channel 5a. Even more advantageously, the thickness of the covering layer 8 is chosen so that the distance separating the analysis wall 6 and the bottom of the first analysis chamber 3a is less than 2 mm.

In an advantageous embodiment, the groove formed by the channel 5a and the analysis chamber 3a belong to the same plane from the outlet of the well 2 to the outlet of the analysis chamber 3a.

The cover layer 8 can be transparent to the optical wavelengths in order to be able to observe the cell cultures present in the wells 2 by optical microscopy, such as fluorescence microscopy when the cover layer 8 covers the wells 2.

According to the embodiments, the analysis wall 6 can be deposited directly on the cover layer 8 or these two layers are separated by at least one bonding layer 9. The bonding layer 9 allows a better mechanical connection between the wall of analysis 6 and the cover layer 8.

To form an effective seal, the material forming the cover layer 8 or the bonding layer 9 may be chosen from polyethylene, polytetrafluoroethylene, polyurethane, a glass fabric impregnated with polytetrafluoroethylene, silicone, polydimethylsiloxane. It is also possible to use an acrylic type adhesive. The bonding layer 9 may be formed by a gel, a paste, a foam.

To promote the contact between the liquid coming from the well 2 and the analysis surface, the thickness of the assembly formed by the cover layer 8 and the connecting element 9 must be less than or equal to the difference between the radius or half-width of the analysis chamber 3a and the radius or half-width of the channel 5a.

It is advantageous to provide that the wall which comprises the reactive coating or the wall opposite the wall which comprises the reactive coating is transparent to the analysis signal so as to facilitate the measurement during the reaction. In this way, it is possible to carry out a measurement during the reaction.

It is advantageous to apply a pressure on the removable analysis wall to improve the sealing of the chip 1. The pressure can be applied by a mechanical compression system such as clamps, magnets or electromagnets, a suction, a pneumatic system.

In order to facilitate the analysis of the constituents of the cellular response in the first analysis chamber 3a, the inventors have observed that it is particularly advantageous to use a revelation reagent. The revealing reagent will react with certain constituents emitted by the cell culture in order to fix markers on the constituents which will be fixed on the probes 6a of the first analysis chamber 3a. The developing reagent can also react with the probes themselves to allow, for example, a positive control or internal revelation calibration.

Various embodiments are possible for reacting the cell response components from the well with a revelation reagent.

The cellular response components or biomarkers present in the liquid and coming out of the well 2a are captured by the probes 6a of the analysis surface.

In one configuration, the capture of the biomarkers causes an analysis signal which makes it possible to follow the cellular response. However, in very many cases, the capture of the biomarkers does not cause an analysis signal and a specific revelation reagent is added and used as a biomarker monitoring element characteristic of the cellular response to be followed. In order to improve the monitoring of the cellular response, it is particularly advantageous to introduce the revelation reagent before the liquid is introduced into the analysis chamber 3a, that is to say before the entry of the analysis chamber, either by mixing the revelation reagent with the liquid leaving the well, or by alternating the flow of reagents and liquid leaving the well. Alternatively, the developing reagent is added to the assay surface after separating the assay wall from the remainder of the assay chamber 3a.

It is possible to carry out the analysis of the cellular response directly in the analysis chamber 3a, if the revelatory reagent is fixed on the biomarker itself attached to the probes 6a and induces an analysable signal and provided that at least part of the chamber is transparent to the induced signal. In other cases, it is advantageous to provide an extraction of the analysis wall out of the analysis chamber 3a to analyze the capturing elements 6a.

If the revealing reagent is introduced into the chip, it is advantageous to introduce the revealing reagent after the exit of the well 2a so as not to disturb the cell culture. The chip advantageously comprises an additional channel which is connected to the first outlet channel 5a between the outlet of the first well 2a and the first analysis chamber 3a at a first junction. In this way, the developing reagent can be mixed with the liquid leaving the first well 2a without interfering with the cell culture.

The developing reagent can be contacted with biomarkers by mixing or alternatively supplying the biomarkers and then the developing reagent in the analysis chamber.

In order to allow real-time analysis by microscopy, the wall presenting the analysis surface can be transparent to optical wavelengths. It can also be performed in a non-self-fluorescent material to allow the use of fluorescence microscopy as an analytical technique. Wall analysis is preferably made of glass, plastic or composite material and covered or not with a thin metal layer, polymer or chemical functionalization.

It is still possible to predict that the measurement of the cellular response can not be carried out directly in the analysis chamber 3a. This situation may occur when the wall 6 of the chamber is not transparent to the radiation used to make the measurement or when the radiation used to make the measurement can damage the biomarkers in the chamber 3a so that the first measure distorts the measures to be followed.

In all these cases, it is advantageous to remove the analysis wall 6 which comprises the reactive coating of the chamber 3a, to put a new analysis wall 6 with a reactive coating and to place the reagent coating put in contact with the liquid containing the biomarkers in a measuring device, for example optically, by luminescence, by absorbance or by fluorescence.

Advantageously, the chip comprises an additional channel 4b / 5b connected to the first output channel 5a between the output of the first well 2a and the first analysis chamber 3a. The outlet channel 5a is intended to be fed with an additional stream which is advantageously a liquid which contains a revelation reagent.

As indicated above, the outlet channel 5a can be defined in the substrate A. It is also possible to provide that the walls of the outlet channel 5a are formed partially or completely in the cover layer 8. It may also be for the additional channel 5b.

Advantageously, the additional channel 4b / 5b comprises a second input channel 4b connected to the first output channel 5a via a mechanical energy sink and a second output channel 5b. The first output channel 5a and the second output channel 5b meet before reaching the analysis chamber 3a. Preferably, the mechanical energy sink is configured to define a pressure drop equal to plus or minus 20% to the pressure drop defined by the first well 2a. According to the embodiments, the liquids from the first outlet channel 5a and the second outlet channel 5b are mixed or not.

It is particularly advantageous to provide that the mechanical energy sink is formed by a second well 2b identical to the first well 2a. When the chip 1 comprises first and second input channels 4a and 4b, first and second wells 2a, 2b and first and second output channels 5a and 5b, it is advantageous to provide that the first and second channels of 4a and 4b define a first plane parallel and offset from a second plane defined by the first and second output channels 5a, 5b.

According to a particular embodiment, the microfluidic chip comprises a mixer (not shown) which is configured to mix at least two laminar liquid streams upstream of the mixer and to deliver a laminar flow output mixer. The mixer is advantageously arranged between the outlet of the first well 2a and the inlet of the analysis chamber 3a. The mixer comprises a first inlet fed by the first well 2a and a second inlet fed by the second well 2b or by a separate inlet of the inlet feeding the first well 2a. The mixer is configured to mix two streams present at the input and output at least one output stream. The output stream is the mix of input streams.

As illustrated in FIGS. 1, 2, 3, 4, 5, 6 and 7, the micro-fluidic chip 1 may comprise a plurality of cell culture wells denoted 2a, 2b, 2c and 2d which are connected to one or more chambers of here analyzes two chambers 3a and 3b. The inlet of each well 2 is connected to an inlet channel 4 and the outlet of each well 2 is connected to an outlet channel 5. An analysis chamber 3 can be connected to one or more different wells 2 by means of intermediate of one to several output channels 5. Wells 2 occupying a small area, it is possible to achieve a large amount of wells 2 in a small area which facilitates the realization of a compact analysis device. The miniaturization of wells also allows the multiplication of the number of wells per device and thus the multiplication of the number of biological samples analyzed by analysis.

According to a first particular embodiment illustrated in FIGS. 1 and 5, a microfluidic chip 1 comprises first, second and third input channels 4a, 4b and 4c connected respectively to first, second and third wells 2a, 2b and 2c mounted fluidically in parallel. The first, second and third wells 2a, 2b and 2c are respectively connected to first, second and third output channels 5a, 5b and 5c. In one embodiment, one of the wells may be replaced by an additional channel. In the illustrated embodiment, it is advantageous to provide that the well 2b is replaced by the additional channel.

The first and second output channels 5a and 5b meet before reaching the first analysis chamber 3a. The second and third output channels 5b and 5c meet before reaching the second analysis chamber 3b. The two analysis chambers are fluidly mounted in parallel between the wells 2a, 2b and 2c and an evacuation 7.

In the embodiment illustrated in FIGS. 1 and 5, the three output channels are connected to the same transverse channel from which the inputs of the two analysis chambers 3a and 3b leave. The first analysis chamber 3a is connected to receive the liquid from the first outlet channel 5a and the liquid from the second outlet channel 5b. The second analysis chamber 3b is connected to receive the liquid from the third outlet channel 5c and the liquid from the second outlet channel 5b. In this configuration, when the pressure applied in the three wells 2a, 2b and 2c is identical, the first chamber 3a receives a stream from the first well 2a and a stream from the second well 2b. The second chamber 3b receives a stream from the third well 2c and a stream from the second well 2b. The information contained in the second output channel 5b is present in both chambers connected in parallel.

In the example illustrated, the two analysis chambers 3a and 3b have an outlet which is finally connected to the evacuation 7.

The analysis chambers 3 and the evacuation 7 out of the substrate are advantageously located in two different parallel planes.

According to an alternative embodiment of the micro-fluidic chip which is represented in FIGS. 2, 3, 4, 6 and 7, a chip 1 can comprise four inputs 4a, 4b, 4c and 4d respectively connected to four wells 2a, 2b, 2c and 2d of cell culture. The chip 1 also comprises two analysis chambers 3a and 3b each connected to two wells 2 of cell culture. The first and second wells 2a and 2b are connected to the first analysis chamber 3a while the third and fourth wells 2c and 2d are connected to the second analysis chamber 3b. Again, one or two wells can be replaced with additional channels to provide reagents.

The two analysis chambers 3a / 3b are finally connected to an evacuation 7 to allow the evacuation of the analyzed products. In the illustrated embodiment, the evacuation 7 is common.

In the embodiments illustrated in the various figures, the microfluidic chip 1 comprises a plurality of wells 2 which are each connected to an input channel 4. Each well 2 has its output channel 5. The output channels 5 of several wells 2 meet before entering an analysis chamber 3. At least one analysis chamber 3 is connected to at least two wells 2 of the microfluidic chip 1.

In the advantageous embodiments illustrated, the outputs of the wells 2 are connected together before reaching the analysis chamber 3 so that the different streams from the plurality of wells 2 enter the analysis chamber 3 by the same inlet. By using a laminar flow in the analysis chamber 3, it is possible to have in the same analysis chamber 3 several liquid flows from several wells 2 without mixing. It is particularly advantageous to have a Reynolds number of less than 100 to further reduce the likelihood of mixing liquids in the analysis chamber. Advantageously, at least two liquids from at least two different wells are present in the analysis chamber.

By judiciously placing probes 6a in several places of the analysis chamber 3 on the analysis surface in a direction perpendicular to the flow of liquid inside the chamber 3, it is possible to analyze the various biomarkers resulting from the Well 2. The different biomarkers are present at the same time in the analysis chamber 3 and are separated from each other in a direction perpendicular to the axis that connects the inlet and the outlet of the chamber 3, that is, that is to say in a direction perpendicular to the axis of circulation of the liquid in the analysis chamber 3. It is particularly advantageous to place several probes 6a on the analysis surface in a direction perpendicular to the direction of flow in the analysis chamber 3 to analyze separately the different cellular responses.

In this configuration, it is possible to perform the analysis of several cell samples placed in different wells 2 with a single analysis chamber 3 without the cellular responses are mixed. Each flow of fluid flows from the inlet to the outlet and carries biomarkers characteristic of the response of the cell sample of a well 2.

The analysis surface is analyzed by a measuring device which can then measure the different cellular responses on the same analysis surface.

Alternatively, it is also possible to have an analysis chamber connected to a single well itself fed by a single input channel. Particularly advantageously, the inlet channels 4 are closed by a septum. In this embodiment not illustrated, the first inlet channel 4a has a first end which opens into the first well 2a and a second end which is closed by a sealed membrane. The waterproof membrane is configured to form a septum which can be pierced for example by a needle. When the needle is removed, the membrane is sealed again. This configuration makes it possible to maintain the tightness of the first inlet 4a and thus to avoid contaminating the first well 2a. The sterility of the cell culture can be maintained despite multiple steps of connection / disconnection of the chip 1 with a flow generator. The seal in the rest of the chip can be obtained by other means.

A waterproof membrane may also be used as a septum at the outlet (s) 7. In this case, one or more needles may be used to pass through the septum and provide fluid communication with a reservoir that recovers the liquids.

When the inlets and outlets are closed by a septum, the microfluidic chip is sealed. This configuration makes it possible to safely move the culture zone and the analysis zone that form a monoblock element.

The flow generator makes it possible to inject one or more liquids into the different inputs of the chip 1 by means of needles which pass through each septum.

The microfluidic chip 1 is advantageously associated with a flow generator, for example a pump, to form a lab on a chip. The pump is preferably configured so that the liquid flows in the analysis chambers 3 are laminar. Advantageously, the liquid flows between the outlet of the well 2 and the inlet in the analysis chamber 3 are also laminar. Preferably, the flow generator is configured so that the flows of liquid between the outlet of the well 2 and the outlet of the analysis chamber 3 have a number of Reynolds less than 100. It is even more advantageous to provide that the flow in the wells 2 is also laminar.

To ensure efficient transport of the elements present in the liquid from the wells 2 to the analysis surface, the fluid flow conditions are configured to promote convective rather than diffused liquid transport of the liquids. It is advantageous to choose flow conditions so that the number of Peclet in the pipe connecting the well 2 to the analysis chamber 3 and preferably in the analysis chamber 3 is less than 1. The number of Peclet is a dimensionless number that represents the ratio of the characteristic time of convective transfer to the characteristic time of diffusion transfer.

The lab on a chip is advantageously configured so that the liquid flow rate is between 0.1 .mu.l / min and 2 ml / min, which allows a laminar flow in the analysis chamber (s) 3. By using such a flow, the fixation of the biomarkers on the probes is better carried out. Preferably, the liquid flow rate is between 0.1 / L / min and 1 mL / min. The value of the liquid flow advantageously corresponds to an average value, for example a mean value over one minute. It is also possible to use a liquid flow in the form of pulses whose flow rate value is between 1 ml / min and 5 ml / min, preferably with a value greater than 4 ml / min. The pulses have durations of less than 10 seconds.

The markers characteristic of the modification of the response of the cells, over time, are found in the flow of liquid leaving the well 2 and entering the analysis chamber 3. With such a flow, the modification of the response of the cells cells can be monitored in real time in the analysis chamber 3 and in particular between the output and the inlet of the analysis chamber 3.

The flow generator is connected to the chip 1 in order to pass a fluid from the well 2 to the analysis chamber 3. The flow generator can be a controller pressure, a syringe pump or a pump (not shown). The flow generator may be configured to supply the chip continuously with fluid or else periodically or aperiodically.

In the context of a continuous feed, a liquid flow is present continuously through the well 2a and the analysis chamber 3a. The flow is non-zero and can be variable in time. The flow can evolve periodically or aperiodically. In the other embodiments, the bit rate changes with a zero rate period and a non-zero rate period. During the non-zero rate period, the rate may be constant or variable.

The flow generator is configured to deliver at least one liquid selected from a feed liquid comprising nutrients, a growth liquid, a test molecule for example a chemical or biological stimuli which may be a drug. It is still possible to provide revelatory reagents. The molecules to be tested may for example be chemical or biochemical molecules, so as to observe the impact of these molecules on the cellular response. The revealing reagents can for example be fluorescent markers in order to use fluorescence microscopy during analyzes.

In a particular embodiment, the well 2a is fed continuously with a first liquid stream. The first stream of liquid may contain nutrients. The first liquid flow may be completed by a second liquid flow whose composition differs from the first liquid flow.

When the composition of the liquid changes over time at the entrance of the well 2a, it is possible to provide that the first flow comprises, during a first period, a flow comprising predominantly nutrients. During a second period, the flow comprises molecules to be analyzed. Preferably, the test molecules are made by means of a liquid which contains a culture medium. The culture medium contains nutrients, but it may also contain salts and buffers. It is also possible to predict that the culture medium contains predefined reagents if it is desired to directly analyze the cells. The liquid flow rate can be constant regardless of the composition of the liquid.

Feeding cell cultures with a continuous flow improves the nutrition of cell cultures relative to a static nutritional intake. Experimental conditions are therefore approaching in vivo conditions.

It is also possible to vary the concentration of biological or chemical stimuli to simulate the conditions of cellular exposure following the administration of a drug or other product. The concentration of stimuli can increase during a first phase then decrease during a second phase.

Cell cultures are subjected to chemical, biochemical, physical or mechanical stimuli in order to evaluate their response, that is, their viability or mortality as well as the nature of the molecules or biomarkers they secrete. In the study of a mechanical stimulus, the pressure and / or the flow rate can change over time and the chemical composition is constant or variable.

The lab-on-a-chip is therefore very interesting because it allows real-time analysis of cell secretions and allows real kinetic studies, close to in vivo conditions. Moreover, the large area of the analysis chamber 3a makes it possible to detect a large number of biomarkers at a lower cost.

This type of lab-on-a-chip is particularly advantageous because it makes it possible to use a large number of different analysis methods. In the case where the chip comprises several wells connected in parallel, it is possible to independently choose the supply of the different wells in the composition, flow and time revolution of these two parameters. The analyzes can be done in parallel which saves time.

It is possible to feed all the wells with the same solution. Alternatively, it is possible to dissociate the fluid feeding the wells between at least two wells so as to compare the cell reaction between the wells. For example, it is possible to compare the cellular response between a flow comprising chemical and / or biological stimuli and a flux devoid of chemical and / or biological stimuli. It is still possible to change the concentration of nutrients, the concentration of chemical and / or biological stimuli between the wells to determine the influence of these different parameters on the cellular response.

In general, the method for analyzing the cellular response of a biological sample can be as follows.

The method comprises a first step of providing a microfluidic chip 1. It is possible to use the chip described above, but it is also possible to use a different chip.

The microfluidic chip 1 comprises a first well 2a filled with a cell sample emitting a first cellular response. The cell sample can be manually deposited, using a pipette or micropipette, in the first well 2a.

The chip further comprises a first input channel 4a for supplying the first well 2a. The first inlet channel opening into an inlet of the first well 2a.

The chip also comprises a first analysis chamber 3a having a first analysis input fluidly connected to the first well 2a by means of a first outlet channel 5a and an evacuation outlet opening into the first analysis chamber 3a and distinct from the first analysis input.

The chip also comprises capture elements 6a disposed on a wall of the first analysis chamber 3, the capture elements 6a being configured to capture at least one biomarker representative of the first cellular response flowing from the first well 2a to the discharge outlet of the first analysis chamber 3a.

The method further includes providing a flow generator configured to apply a first liquid flow supplying the first well 2a so that the first cellular response is taken from the first well 2a to the discharge outlet through the first analysis chamber 3a. Then, the biomarker is moved from the first well 2a to the evacuation outlet of the first analysis chamber so that the at least one biomarker is captured by the capturing elements 6a. the capturing elements 6a are analyzed to follow the cellular response of the cell sample.

The flow generator is configured to apply liquid streams to at least the first input channel 4a.

Advantageously, the well is filled with a liquid that contains nutrients before introducing the cell culture into the well. In a particular embodiment, the input channels 4 of the chip are connected to the flow generator and the flow generator is configured so that a liquid feeds the well 2 with a first liquid which advantageously comprises nutrients. In a particular mode of organization, the flow generator is configured to completely fill the well without filling the analysis chamber. Advantageously, the generator is configured to fill a plurality of wells with the same flow rate for each well.

Once the well is filled with a liquid, a cell culture is introduced into the well. For example, a cell culture matrix is introduced into the well. It is particularly advantageous to introduce the cell culture into the well after having filled the well with a fluid which avoids leaving the cell culture in the open air and therefore drying out of the cell culture. Advantageously, at least the channels 4 are filled before introducing the cell culture into the wells. In one case, the closing of the well 2 is carried out simultaneously with the introduction of a first sealing element around the analysis chamber 3. The sealing element leaves the analysis chamber 3 accessible. .

In a particular method of use, the closure of the well 2 is carried out before the introduction of the cells in the well 2. The cells will then be placed in the well 2 by injection by means of the inlet channel 4 or through the cover layer 8, for example, through a syringe.

The analysis chamber 3 can then be closed again by means of the analysis wall 6. Once the analysis chamber 3 is sealed, the flow generator can fill the analysis chamber 3 with the liquid. Preferably, the flow generator fills the analysis chamber 3 with a flow coming from the well 2. Advantageously, the flow generator is configured to fill the well 2 and then the analysis chamber 3 until evacuation 7.

The analysis method advantageously comprises a step of feeding the first well 2a comprising the cell culture with a first liquid having a first composition and comprising nutrients. The first well 2a is fed with a first flow and a first pressure.

In a second step, the first well 2a can be fed with a second liquid comprising a second composition. The first well 2 is fed with a second flow and a second pressure, at least one parameter chosen from the second composition, the second flow and the second pressure being respectively different from the first composition, the first flow and the first pressure. The cell culture emits biomarkers in response to the second liquid. The first and second fluid supply is configured for the biomarkers to reach the analysis chamber 3. The biomarkers are picked up by the probes 6a and it is possible to compare the difference in response between the first liquid and the second liquid for a second time. same cell culture in the same chip. By changing the pressure and / or the flow, it is possible to simulate a mechanical stress on the cell culture. By changing the composition of the nutrient medium, it is possible to simulate chemical stress. However, it is particularly advantageous to add at least one molecule to the second liquid, that is to say the molecule to be studied. Alternatively, the molecule to be studied is present continuously. The constituents of the first liquid and the second can be identical and with different concentrations.

The biomarkers representative of the cellular response contained in the liquid from the cell culture wells are then captured on the probes 6a present in the analysis chamber 3. If necessary, a revelation reagent is brought into contact with the biomarkers captured on the cells. probes 6a to highlight an analyzable signal.

Advantageously, the method comprises the provision of at least one revelation reagent configured to react with the representative biomarker or biomarkers of the first cellular response captured by the capture elements 6a. The developing reagent passes through the first analysis chamber 3a simultaneously or after the passage of the first cellular response in the first analysis chamber 3a. The analysis of the capture elements 6a is carried out by searching for the revealing reagent.

When the microfluidic chip comprises a plurality of wells for example at least two, three or four wells, it is possible to use the input channels of the chip differently.

In one configuration, a first well 2a is filled with cells and a second well 2b is not filled with cells. The first well 2a is subjected to a first flow of liquid containing nutrients and possibly to chemical or biological stimuli. The second well 2b then becomes an additional input which opens into the first output channel 5a by a first junction located between the first well 2a and the first analysis chamber 3a. This is subjected to a second flow of revealing reagents. The second stream enters the analysis chamber 3 without being in contact with the cell sample.

So that the analysis chamber 3a is fed both with reagents from the additional inlet or the well 2b and the liquid from the cell culture well 2a, different embodiments are possible. The outlets of the first well 2a and the second well 2b meet at the first junction in a mixer so that the second stream of reagents mixes with the first biomarker stream in the outlet channel 5a after the first join. At the outlet, the mixer delivers a liquid corresponding to the mixture of the two flows. There is then no risk that the reactants or the injection rate of the reagents disturb the cells of the first well 2a.

Alternatively, the analysis chamber is fed alternately with the well 2a and then with the well 2b so that all the probes alternately see the liquid containing the biomarkers of the cellular response and the liquid containing the revealing reagents.

For example, if the chip has three wells as illustrated in FIGS. 1 and 5, it is possible to associate a well and its input channel with a flow of reagent. The other two wells are filled with cell cultures. Outputs from the wells containing cell cultures each join with the exit of the well associated with the revealing reagent. Again, it is possible to provide that several wells contain cells and that these wells are fed with different compositions. The flow of reagents is introduced into the central well and the end wells comprise the cell cultures.

The well containing the cell culture is fed with a nutrient liquid. After a first waiting time, the cell culture is also fed by the molecule to be studied. Simultaneously with the injection of the molecule to be studied or after the injection, the revelation reagent is applied on the second inlet channel so that the revealing reagent is introduced into the analysis chamber by mixing or succession with the liquid from the cell culture wells. Advantageously, this sequence of steps is performed by the flow generator which comprises a control circuit. This configuration is particularly advantageous when the revelation reagent is detrimental to the cell culture.

In another embodiment, both wells 2a and 2b are filled with cell cultures. As before, a flow of liquid containing nutrients is applied in each of the wells 2. The flow of liquid from at least one of the wells 2 is then modified in order to apply a molecule to be studied in each of the wells. It is conceivable to apply the molecule to be studied in one well.

The developing reagent may be applied to the assay surface off the chip after removal of the assay wall. This embodiment is particularly advantageous in combination with the chip illustrated in Figure 2 where two wells are used to feed the same analysis chamber. The embodiment illustrated in FIGS. 1 and 5 makes it possible to compare different conditions by using the condition applied in the well 2b as a reference.

Well 2b can also be advantageously associated with the supply of reagent, wells 2a and 2c being then used to study operating conditions for stimulating cell cultures. Each analysis chamber receives the same reagent and a specific cellular response from well 2a or well 2c. Alternatively, the well 2b receives the cell culture and the wells 2a and 2c are associated with different reagents and / or the two analysis chambers have different probes 6a.

The chip also makes it possible to provide two different cell cultures in two different wells whose outlets join or not in the same analysis chamber. Both wells are subjected to the same liquid flow, preferably simultaneously. It is then possible to observe the response of the two different cultures.

It is still possible to provide for two wells to be filled by two different cultures and for each culture to be subjected to different liquid flows. Integration on the same chip makes it possible to ensure that the other conditions studied are identical for example with regard to temperature, humidity and / or the atmosphere. In an advantageous embodiment, the well is maintained at a constant temperature. setpoint temperature, for example by means of an incubator. The set temperature is advantageously between 20 and 45 ° C, preferably equal to 37 ° C. In a particular embodiment, the incubator is configured to apply the set temperature during filling of the well and during filling of the analysis chamber. Alternatively, the incubator is configured to apply the set temperature once the cell culture is introduced into the well. The incubator may also be configured to provide a controlled atmosphere with 5% vol of CO2 and at least 90% by volume of moisture. In order to have study conditions closest to in vivo conditions, it is particularly advantageous to provide for the flow generator to feed the well and the analysis chamber when the chip is at the set temperature.

Claims

claims

A micro-fluidic chip (1) comprising a substrate (A) defining:

A first well (2a) intended to receive a biological sample, the substrate (A) defining side walls and at least one bottom or top wall of the first well (2a), the first well (2a) opening on the surface; one side of the substrate (A), the first well (2a) being closed by means of a covering layer (8),

A first inlet channel (4a) intended to feed the first well (2a) with a first liquid, the first inlet channel (4a) terminating in a first inlet opening into the first well (2a),

A first outlet channel (5a) having a first end formed by a first outlet of the first well (2a) for discharging the first liquid,

A first analysis chamber (3a) having a first analysis input connected to the first well (2a) by means of the first output channel (5a) and an evacuation outlet opening into the first analysis chamber (3a). ) and distinct from the first analysis input,

micro-fluidic chip in which the first inlet and the first outlet of the first well (2a) define a different first axis and not parallel to a second axis defined by the first analysis input and the discharge outlet of the first chamber; analysis (3a),

characterized in that the first analysis chamber (3a) is closed by an analysis wall (6) comprising capture elements (6a) configured to capture at least one biomarker of the first liquid flowing in the first analysis chamber (3a), the analysis wall (6) being removably mounted relative to the substrate (A) and with respect to the covering layer (8) so as to be able to dismount the analysis wall (6) independently of the cover layer (8).

2. micro-fluidic chip (1) according to claim 1, wherein the cover layer (8) is removably mounted relative to the face of the substrate (A).

3. micro-fluidic chip (1) according to one of claims 1 and 2, wherein the cover layer (8) defines at least one through hole forming a ring surrounding the outlet of the first outlet channel (5a), defined ring in the layer of cover (8) forming completely or partially the side walls of the first analysis chamber (3a), the face of the substrate (A) forming a bottom wall of the first analysis chamber (3a).

4. Microfluidic chip (1) according to one of claims 1 to 3, wherein the side walls of the first analysis chamber (3a) are partially formed in the substrate (A), the first analysis chamber (3a) being formed by a first cavity opening on the face of the substrate (A), the covering layer (8) defining a ring surrounding the first cavity and partially forming the side walls of the first analysis chamber (3a).

5. micro-fluidic chip (1) according to one of the preceding claims wherein the side walls of the first outlet channel (5a) are formed by a groove in the face of the substrate (A), the cover layer (8). closing the first well (2a) and the first outlet channel (5a) to the first analysis chamber (3a).

6. Microfluidic chip (1) according to one of the preceding claims wherein the first outlet channel (5a) is defined by a groove formed in the cover layer (8) which closes the first well (2a).

A micro-fluidic chip (1) according to any one of the preceding claims comprising an additional channel (4b, 5b) connected to the first outlet channel (5a) between the outlet of the first well (2a) and the first chamber of analysis (3a), the additional channel (4b, 5b) being advantageously formed by an additional groove in the face of the substrate (A) and / or in the cover layer (8).

8. Microfluidic chip (1) according to any one of the preceding claims wherein the first analysis chamber (3a) comprises first and second analysis inputs respectively defining first and second directions of introduction of liquid in the first analysis chamber (3a).

9. micro-fluidic chip (1) according to any one of the preceding claims having an evacuation (7) connected to the discharge outlet of the first analysis chamber (3a) and the first and second septa configured to close the sealing the first inlet channel (4a) and the outlet (7).

10. Microfluidic chip (1) according to any one of the preceding claims wherein the distance separating the analysis wall (6) and the bottom of the first analysis chamber (3a) is less than 2 mm.

11. Laboratory (20) on micro-fluidic chip comprising:

A microfluidic chip (1) according to any one of the preceding claims, the micro-fluidic chip comprising an analysis wall (6) removably closing the first analysis chamber (3a), the wall of analysis (6) being provided with capturing elements (6a) configured to capture at least one biomarker of the liquid flowing in the first analysis chamber (3a) and from the first well (2a);

A flux generator connected to the first input channel (4a) of the microfluidic chip (1), the flux generator being configured to emit at least a first liquid flow intended to pass through at least the first well (2a ) filled with a cell sample and the analysis chamber (3), the first liquid flow having a flow rate between 0.1 / L / min and 2 mL / min so that the flow of liquid flowing in the first analysis chamber (3a) is laminar in at least half of the first analysis chamber (3a) having the discharge outlet.

12. Laboratory (20) micro-fluidic chip according to the preceding claim wherein the flow generator is connected to an additional channel (4b) of the microfluidic chip and wherein the flow generator is configured to:

Applying a first flow on the first inlet channel (4a) so as to feed the first well (2a) during a first period, the first flow comprising a first liquid comprising at least nutrients,

Applying an additional flow on the additional channel (4b) comprising reagents configured to react with biomarkers characteristic of a cellular response of the biological sample contained in the first well (2a), the capturing elements (6a) being configured to capture and / or react with biomarkers.

13. Process for producing a micro-fluidic chip comprising the following successive steps:

supplying a microfluidic chip (1) according to claim 1, - closing the first well (2a) by means of a cover layer (8) which at least partially forms the side walls of the first analysis chamber (3a),

- Close the first analysis chamber (3a) by means of an analysis wall (6) comprising capturing elements (6a) configured to capture biomarkers of the liquid flowing in the first analysis chamber (3a).

14. A method of analyzing a cell sample comprising:

Providing a microfluidic chip (1) comprising:

a first well (2a) filled with a cell sample emitting a first cellular response containing at least one biomarker;

a first analysis chamber (3a) devoid of a cell sample and having a first analysis input fluidly connected to the first well (2a) by means of a first outlet channel (5a) and an evacuation outlet opening in the first analysis chamber (3a) and separate from the first analysis input,

o capture elements (6a) disposed on a wall of the first analysis chamber (3), the capture elements (6a) being configured to capture said at least one biomarker flowing from the first well (2a) to the first analysis chamber (3a);

Applying a first stream of liquid feeding the first well (2a) so that the first cellular response is taken from the first well (2) to the discharge outlet through the first analysis chamber (3a), said at least one biomarker flowing in the first analysis chamber to be picked up by the capturing elements (6a),

• analyze the capturing elements (6a),

wherein a one-piece substrate (A) at least partially defines the first well (2a) and wherein the pick-up members (6a) are removably mounted relative to the first analyzing chamber (3a).

15. Analysis method according to the preceding claim comprising the provision of at least one developing reagent configured to react with said at least one biomarker representative of the first cellular response captured by the capture elements (6a), said at least one revealing reagent crossing the first analysis chamber (3a) simultaneously or after the passage of said at least one biomarker in the first analysis chamber (3a), the analysis of the capture elements (6a) being performed by searching for said at least one revelation reagent.

16. Analysis method according to the preceding claim comprising applying a second stream to an additional input of the microfluidic chip (1), the additional input opening into the first output channel (5a) through a first junction located between the first well (2a) and the first analysis chamber (3a) so that the second flow enters the first analysis chamber (3a), the second flow comprising the at least one revelation reagent so that least a revealing reagent is devoid of contact with the cell sample.

17. Analysis method according to the preceding claim comprising the filling of the first output channel (5a), after the first junction, alternatively by the first stream comprising the said at least one biomarker and the second stream comprising the said at least one reagent of revelation.

18. Analysis method according to claim 16 wherein the first junction is associated with a mixer configured to mix the first stream with the second stream in the first output channel (5a) after the first junction.

19. Analysis method according to any one of claims 14 to 18, wherein the microfluidic chip (1) comprises a second well (2b) comprising an additional cell sample emitting a second cellular response comprising an additional biomarker, the second well (2b) being fluidly connected to the first analysis chamber (3a) by a second output channel (5b) joining the first output channel (5a) before the first analysis chamber (3a), a generator feeds the first and second wells (2a, 2b) to take the representative biomarker and additional biomarker respectively of the first and second cellular responses in the first analysis chamber (3a) and deliver first and second laminar flows to the first and second the interior of the first analysis chamber (3a), the biomarker and the additional biomarker being respectively present in the first and second laminar flows.

20. Analysis method according to any one of claims 14 to 19, wherein the first well (2a) is filled by the cell sample before the closure of the first well (2a) by a cover layer (8), the cover layer (8) at least partially forming the side walls of the first analysis chamber (3a).

21. Analysis method according to any one of claims 14 to 20, wherein the first analysis chamber (3a) is closed by means of an analysis wall (6) comprising the capturing elements (6a). .

22. The analysis method according to the preceding claim, comprising dismantling the analysis wall (6) to perform the analysis of the capturing elements (6a) outside the first analysis chamber (3a).

23. Analysis method according to the preceding claim, wherein the chip (1) is defined in a substrate (A), the substrate (A) defining a bottom and a side wall of the first well (2) and a groove forming the first output channel (5a) and wherein the analysis method comprises sealing the first well (2) and the first output channel (5a) in a sealed manner by means of a removable cover layer (8).

24. Analysis method according to the preceding claim, wherein the cover layer (8) defines an opening (8a) forming a closed ring around the first analysis chamber (3a) and wherein the method comprises, after the installing the cover layer (8), installing an analysis surface (6a) sealing the first analysis chamber (3a) in a sealed manner, the analysis surface (6) comprising the capture (6a).

25. Analysis method according to any one of claims 22 to 24, wherein the side walls of the first outlet channel (5a) are at least partially formed in the cover layer (8).