EP2699353A1

EP2699353A1 - Microfluidic system for controlling the concentration of molecules for stimulating a target

Info

Publication number: EP2699353A1
Application number: EP12720642.3A
Authority: EP
Inventors: Maxime DAHAN; Mathieu Morel; Jean-Christophe Galas; Denis Bartolo; Vincent Studer
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite Victor Segalen Bordeaux 2; Universite Pierre et Marie Curie Paris 6; Ecole Normale Superieure; Universite Bordeaux Segalen; Fonds ESPCI Georges Charpak
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite Victor Segalen Bordeaux 2; Universite Pierre et Marie Curie Paris 6; Ecole Normale Superieure; Universite Bordeaux Segalen; Fonds ESPCI Georges Charpak
Priority date: 2011-04-22
Filing date: 2012-04-20
Publication date: 2014-02-26
Also published as: JP6140684B2; JP2014518509A; FR2974360A1; WO2012143908A1; FR2974360B1; RU2013151882A; AU2012245910A1; MX2013012365A; CN103842084B; RU2603473C2; AU2012245910A8; KR20140063521A; AU2012245910B2; US20140113366A1; US9164083B2; CN103842084A; CA2833857A1

Abstract

The invention relates to a microfluidic system for controlling a card for the concentration of molecules capable of stimulating a target, for example formed by an assembly of living cells, characterized in that the system comprises a microfluidic device (1) comprising: n_c ≥ 1 microfluidic channel(s) (4, 40), the or each channel being provided with at least one inlet orifice for at least one fluid and with at least one outlet orifice for this fluid; n₀ ≥ 2 openings (47, 470) formed in the microfluidic channel or distributed in the various microfluidic channels, said openings being arranged in one and the same plane so that they form a network having at least one dimension in this plane, the numbers n_c of microfluidic channel(s) and n₀ of openings being linked by the relationship (I) with 1 ≤ i ≤ n_c and n_0/ci the number of openings for the channel c_i ; at least one microporous membrane (5) covering the network of openings, the target being intended to be positioned on the side of the membrane which is opposite the microfluidic channel(s); one or more fluid feed means for feeding the or each microfluidic channel with fluid, at least one of these fluids comprising molecules for stimulating the target.

Description

MICROFLUIDIC SYSTEM FOR CONTROLLING THE CONCENTRATION OF STIMULATION MOLECULES OF A TARGET

The present invention relates to the field of microfluidics.

Microfluidics implements systems of micrometric dimensions, the size of which is generally between a few tens and a few hundred microns.

These systems find application in many fields such as cell diagnostic tests, drug development, basic biology or cosmetology.

In these fields, there is a growing demand for microfluidic systems to quantitatively determine the response of living cells to certain molecules and particularly, the response to a spatially and temporally controlled concentration map.

For example, it may be to measure the response of cancer cells to molecules used for chemotherapy. To precisely determine this response, it is necessary to exercise control over the application of the molecules that will generate this response. This control can concern the quantity of molecules interacting with the cancer cells, the concentration map of the molecules to which the cancer cells are subjected, the evolution over time of the quantity of these molecules and / or the concentration map of these molecules. applied to cancer cells, etc.

In the field of cosmetology, microfluidic systems can be used for toxicity tests of certain molecules on living cells and / or cellular tissues. The control of the amount of molecules, possibly toxic, administered to the cells and the way in which these molecules are administered is necessary to determine the threshold of toxicity. An example of a microfluidic system widely used to stimulate living cells is presented in document US Pat. No. 7,374,906. This microfluidic system makes it possible, in particular, to subject living cells to a concentration gradient of molecules, whose map is here a linear, stable profile. in time.

A major disadvantage of this type of microfluidic system is that living cells are subjected to a flow generating disturbing shear forces. This shearing effect is particularly troublesome when one seeks to study the chemotactic response of the growth cone of nerve cells. Indeed, the flow generates shear stresses that modify the response of the target cells in the best case, or even cause the death or tearing of the cells.

The physiological behavior of the living cells thus studied is disturbed with the system disclosed in this document.

Solutions have therefore been proposed to subject living cells to a map of concentration of molecules and / or combinations of several maps of concentration of different molecules, without being disturbed by a flow.

Such a microfluidic system is for example presented in the article "Microfluidic device for the combinatorial application and maintenance of dynamically imposed diffusional gradients", R. L. Smith et al., (2010) 9: 613-622.

This microfluidic system comprises a microfluidic device 100 and means (not shown) for supplying the device with fluids.

The microfluidic device 100 disclosed herein is shown in FIG. 1 in an exploded perspective view.

It comprises a PDMS structure in which several fluid supply channels 130a, 130b, 130c, 130d are formed independent of one another. The upper wall 120 of each of these channels 130a, 130b, 130c, 130d has one or more orifice (s) 110 passing through this wall. On the side of the wall 120 opposite the microfluidic channels, there is a culture chamber 140 for living cells filled with a culture medium 150 in the form of a gel, such as agarose. The wall 120 thus forms a membrane, insofar as it makes it possible to separate two media, namely the microfluidic channels 130a, 130b, 130c, 130d, in which a fluid is intended to circulate, and the culture chamber 140.

A glass plate 160 makes it possible to close the culture chamber 140, in its upper part.

The fluid supply means are not shown. It should however be noted that one or more fluid (s) can be introduced into each of the channels 130a, 130b, 130c, 130d, these fluids comprising molecules for stimulating living cells, passing through the wall 120 PDMS via the orifices of diffusion 110.

To control the culture of living cells in space, it is possible, with the microfluidic device 100, to choose the channels in which a fluid is sent with molecules for stimulating living cells. It is thus possible to choose precisely the orifices 110 from which these molecules will diffuse into the culture chamber 140.

Moreover, to control the culture of living cells over time, it is possible to shift the fluid supply of the various channels 130a, 130b, 130c, 130d over time.

The device 100 also has a great flexibility in the choice of the stimulation molecules that can be used, insofar as each of the fluid supply channels 130a, 130b, 130c, 130d provides a means of supply that is specific to it.

This microfluidic system however has several disadvantages. It requires the use of a culture chamber 140 comprising a culture medium 150 in the form of a gel to prevent the passage of fluid from the fluid supply channels to the culture chamber.

In the case in point, the device actually manufactured and tested has square orifices of 20μΐη x 20μΐη. These dimensions are relatively large and promote the passage of the fluid to the culture chamber 140.

The authors specify that 110 square holes of smaller dimensions, for example 4um x 4μιη, could be considered. However, the manufacturing technique used (DRIE) is known to not allow the formation of orifices below a maximum aspect ratio, typically 1: 20, this aspect ratio being here defined by the ratio of the dimension of the side of the orifice to the depth of this orifice. For a given dimension on the side of an orifice 110, this maximum aspect ratio limits the depth of the orifice and therefore the hydraulic resistance that can confer this orifice. This increases the risk of fluid passage of a channel 130a, 130b, 130c, 130d to the culture chamber 140.

Whatever the size of the orifices, it is therefore understood that the fluid would pass to the culture chamber 140 in the absence of a culture medium 150 in gel form.

It should also be noted that the indispensable presence of the gel brings disadvantages in the operation of the microfluidic device 100.

Indeed, the gel slows down the diffusion of the stimulation molecules towards the targeted living cells. Thus, the stabilization of the concentration map of these stimulation molecules within the culture chamber 140 is slow.

In addition, it should be noted that the pacing molecules diffuse in all directions in the gel. Consequently, for each orifice 110 taken independently of the others, the stimulation molecules are distributed, at a target located in the gel, over a surface greater than the passage surface of an orifice 110 located in the wall 120. The stimulation of the target by specific molecules is therefore less precise than it is theoretically with a prior choice for the sizing of the orifices. At the level of the target, one therefore loses in spatial resolution, compared to the spatial resolution theoretically provided by the dimensions of an orifice.

In addition, it should be noted that the diffusion orifices 110 have a certain surface density on the surface of the wall 120, which can hardly be increased. In the case in point, the distance between two neighboring orifices made in the PDMS wall 120 is between 300μηι and 400μηη.

However, the density of the orifices 110 that can be obtained with the manufacturing method used in this article is limited. Indeed, the DRIE method used in this article to make the microfluidic device has limits on the density of microfluidic channels that can be obtained. Each of these microfluidic channels opening, by design, on a single orifice 110, it follows that the surface density of the orifices 110 is consequently also limited.

This disadvantage is added to the fact that the accuracy of the stimulation of a target theoretically provided by the dimensions of an orifice is not that which is actually obtained at the target.

An object of the invention is to overcome at least one of these disadvantages.

To achieve this objective, the invention proposes a microfluidic system for controlling a concentration map of molecules capable of stimulating a target, for example formed by a set of living cells, characterized in that the system comprises:

a microfluidic device comprising: • n _c ≥ 1 channel (aux) microfluidic (s), the or each channel being provided with at least one inlet for at least one fluid and at least one outlet for this fluid;

• n ₀ ≥ 2 openings formed in the microfluidic channel or distributed in the different microfluidic channels, said openings being arranged in the same plane so that they form a network in this plane, the numbers n _c of microfluidic channel (ux) ( s) and n ₀ openings being connected by the relation n ₀ = Σ ^ τΐο ^. with 1 <i≤ n _c and n _{0 / c} . the number of openings for the channel C _t (the term Σ corresponds to the operator "sum");

At least one microporous membrane covering the network of openings, the target being intended to be disposed on the side of the membrane which is opposite to the microfluidic channel (s);

one or more fluid supply means for supplying the or each microfluidic channel with fluid, at least one of these fluids comprising molecules for stimulating the target.

The system may provide other technical features, taken alone or in combination:

- The microporous membrane is provided with pores whose hydraulic diameter is between Ο, Οδμητι and 12μηπ, preferably between Ο, Οδμΐη and 3μηι;

- the surface density of the pores of the microporous membrane is between 10 ³ and 10 ¹⁰ pores / cm ^2;

the microporous membrane is made of a material chosen from: glass, polycarbonate, polyester, polyethylene terephthalate, quartz, silicon, silica or silicon carbide;

a cover is provided for the microfluidic channels, said cover being made of a material chosen from: glass or silicon, a non-elastomeric photocured polymer, a metal, an electrically or semiconducting conducting alloy, a ceramic, quartz sapphire, an elastomer; said at least one inlet orifice and said at least one outlet orifice for the fluids are formed in the lid;

the microfluidic channels each comprise at least one photocured and / or thermoset resin wall;

- There is provided a culture chamber of said target, closed, or a microfluidic channel, disposed on the side of the microporous membrane which is opposite the (x) channel (s) microfluidic (s), the target thus being located in the chamber or said another microfluidic channel;

the chamber or the microfluidic channel comprises a base made of an optically transparent material, this base being disposed on the other side of the chamber or the microfluidic channel, with respect to the microporous membrane;

the microfluidic chamber or channel comprises side walls made of photocured and / or thermoset resin;

a plurality of means is provided for feeding the microfluidic channels, each of these supply means feeding one of the microfluidic channels;

an optical display means is provided;

the optical display means uses a photoactivation localization microscopy technique or a stimulated emission depletion microscopy technique;

the openings form a two-dimensional network in the plane to which they belong;

- The respective centers of two adjacent openings are separated by a distance between 10μΐη and 250μηι.

Other features, objects and advantages of the invention will be set forth in the following detailed description with reference to the following figures:

- Figure 2 is a diagram of a microfluidic device according to the invention, in a partially cut perspective view; FIGS. 3 (a) to 3 (d) represent, as the case may be, steps of a process for manufacturing the microfluidic device represented in FIG. 2 or intermediate structures obtained at the end of certain steps of this process;

FIGS. 4 (a) to 4 (c) show intermediate structures obtained during the manufacture of an assembly formed by a base and side walls of the device, said assembly being intended to form a part of the microfluidic device of FIG. 2;

FIG. 5 (a) represents fluids flowing in the microfluidic channels of the microfluidic device according to the invention represented in FIG. 2, one of these fluids comprising stimulation molecules for the target cells and FIG. b) represents a concentration profile of stimulation molecules in a chamber of the device of FIG. 2;

- Figure 6 shows another microfluidic device according to the invention, in a sectional view;

FIGS. 7 (a), 7 (b), 7 '(a), 7' (b) and 7 (c) to 7 (f) represent manufacturing steps of the microfluidic device of FIG. 6 or intermediate structures obtained in the manufacture of this microfluidic device;

FIG. 8 is a diagram showing the microfluidic device of FIG. 6, in a partial bottom view;

FIG. 9 represents, in a perspective view, the microfluidic channels of the microfluidic device of FIG. 6, which are arranged such that each microfluidic channel comprises several openings;

- Figure 10 shows, in a view from above, microfluidic channels of a microfluidic device according to a variant of Figure 6, these channels being arranged such that each microfluidic channel comprises an opening opening on a microporous membrane of this device;

- Figure 11 shows a manufacturing step of the device shown in Figure 10; FIGS. 12 (a) to 12 (d) represent, as the case may be, steps of a method of manufacturing an alternative embodiment of a microfluidic device according to the invention or intermediate structures obtained at the after certain steps of this process.

First of all, we present in support of FIG. 2, a microfluidic device 1 according to the invention comprising two microfluidic channels, each of these channels being provided with openings opening onto the membrane, as well as its manufacturing method. in support of Figures 3 (a) to 3 (d) and 4 (a) to 4 (c).

This microfluidic device 1 comprises a lid 2, advantageously rigid, provided with orifices 21, 22 for the circulation of fluids in microfluidic channels, a side wall 3 and a central wall 30, both advantageously made of photocuric and / or thermoset resin . In particular, the side wall 3 of the device 1 is made of a single layer of photocured resin and / or thermoset.

The microfluidic device 1 also comprises, in its lower part, two openings 47, 470 covered by a microporous membrane 5 extending transversely to the base of the side wall 3 and the central wall 30. By opening 47, 470 is meant the end surface of the channel which extends between the walls of the device and which is intended to be covered by the membrane 5.

The openings 47, 470 are arranged in the same plane and can be likened to an array of openings, in this case to a dimension, in this plane. In the context of the invention, the term "network" of openings simply designates the fact that there are several openings, without there necessarily being a link between these openings and / or a specific arrangement of these openings in the plan to which they belong.

For example, in Figure 1, the openings 47, 470 are fed by two different microfluidic channels and necessarily arranged in a row in the same plane. On the other hand, in other embodiments which are described below, certain openings may be fed by the same channel, the openings being also arranged in two dimensions in the same plane.

The walls 3, 30 and the cover 2 make it possible to define two microfluidic channels 4, 40, closed at their respective openings 47, 470 by the microporous membrane 5. The fluid inlet for each of these channels 4, 40 respectively corresponds to at the orifice 21 or 22. The fluid outlets for these microfluidic channels are not shown.

The microporous membrane 5 separates two media, namely the microfluidic channel and the external medium of this channel, this external medium being for example formed by a culture chamber 8 in which the target to be stimulated is intended to be arranged. In this respect, it should be noted that the gel used in the Smith et al. is not a membrane, because it does not separate the microfluidic channel and the culture chamber, but instead forms a culture medium filling the culture chamber.

In addition, the microporous membrane 5 prevents the fluid intended to flow in the microfluidic channels 4, 40 to pass on the other side of this membrane, the latter, however, to broadcast molecules that may stimulate the target, which are likely to be transported by the fluid in at least one of the microfluidic channels 4, 40 as will be detailed in the following description. The device 1 according to the invention does not require the presence of a gel in the culture chamber 8.

The microfluidic device 1 also comprises a base 6, advantageously rigid and transparent, and side walls 7a, 7b, advantageously made of photocured resin and / or thermoset. These lateral walls 7a, 7b, the base 6 and the microporous membrane make it possible to form the chamber 8. In order to form the chamber 8, four side walls are provided, these walls actually being comparable to a single contour, since the manufacturing process advantageously performs these walls in one piece. The bottom of the chamber 8 is formed by the upper face 61 of the base 6, which is intended to receive the target, for example formed of living cells. In this case, the living cells are intended to be placed apart from the microporous membrane 5, on the base 6 of the chamber 8. They can thus be cultured under standard conditions, separately from the microfluidic device 1.

The microfluidic channels 4, 40 make it possible to circulate a fluid comprising molecules capable of stimulating the target. This is done, as will be explained in more detail in the following description, by diffusion through the microporous membrane 5 to the chamber 8, then by diffusion through the chamber 8 (culture chamber) at the bottom of which find, for example, living cells (CV) that we try to stimulate.

Advantageously, the base 6 is made of an optically transparent material, for example glass. This is interesting because it is then possible to have an optical display means 18 outside the device for visualizing, for example, the response to a stimulation of the living cells arranged at the bottom of the chamber 8.

The cover 2 may be made of a material chosen from: glass or silicon, a non-elastomeric photocrosslinked polymer, a metal, an electrically conductive or semi-conductive alloy, a ceramic, quartz, sapphire, an elastomer.

The microporous membrane 5 is chosen to avoid any fluid passage between the mircrofluidic channels 4, 40 and the chamber 8. In reality, the microporous membrane 5 can not be completely sealed to a fluid passage. Also, it can be considered that the cells in the chamber 8 are not subject to any flow if the fluid flow rate through the microporous membrane 5 is less than a limit value.

One can for example consider that this limit speed is of the order 1 μΐ ^η / s. In this case, the shear stresses applied to the cells are negligible. Furthermore, the speed in each microfluidic channel 4, 40 may be between ΙΟΟμΐη / s and ΙΟΟΟΟμηΊ / s, or even be greater than 10,000 μηι / s.

Also, to obtain the limit value of Ι μΐη / s, the hydraulic resistance R _h .membrane of the microporous membrane 5 must be, depending on the speed of the fluid in the channel 4, 40 from 100 to 10,000 times greater than the hydraulic resistance Rh channel channel of the microfluidic channel 4, 40.

For example, if the flow velocity of the fluid in the microfluidic channel 4, 40 is ΙΟΟΟΟμίη / s, then the inequality must be respected:

10 000 ^* R _n , channel <Rh.membrane (R1) to ensure that the velocity of the fluid through the membrane 5 is well below the considered limit value of 1 μΐτι / s.

Furthermore, if we consider a rectangular microfluidic channel 4, 40 of height h, width w and length L, and a microporous membrane 5 of thickness e and having identical and cylindrical pores of radius r _p0 re, with a density surface of pores p, then the relation (R1) is written in the form:

10,000 ^* μ.Ι_ / (wh ³ ) <Me / (r _p0 re p Lw) (R2) is: Θ = r _pore ⁴ .p / e <10 ^-4 * h ³ / L ² (R 3)

For a microfluidic channel 4, 40 of height h = 100 μΐΎΐ, width w = 1000 μΐ ^η and length L = 1000 μm, then the term Θ must be less than 10 ^"10 m to respect the relation (R3). In addition, if we consider cylindrical pores with a radius of 1 μΐη and a membrane thickness of 10 μm, then the pore surface density p should be less than 10 ⁵ pores / cm ² . The relation (R3) can of course be generalized as a function of the considered value of the limit velocity of the fluid passing through the microporous membrane 5, on the one hand, and of the flow velocity of this fluid in the microfluidic channel 4, 40 on the other hand.

The microporous membrane 5 may have pores whose hydraulic diameter is between 0.05 μηι βί 12 μηι. In particular, if the pore is cylindrical then, the hydraulic diameter of the pore corresponds to its diameter.

Advantageously, this hydraulic diameter will however be between 0.05 μητι and 3 μηπ. Indeed, it should be noted that the use of a membrane with pores whose hydraulic diameter is less than 3μΐη will avoid any passage of flow in the chamber 8, for most conditions of use likely to be encountered.

Currently, membrane manufacturers offer on the market membranes whose hydraulic pore diameter is generally greater than 0.2 μητι. In the context of the invention, the pores may therefore have hydraulic diameters advantageously between 0.2 μΐη and 3 μηι. However, there is in theory no lower limit for the hydraulic pore diameter, which is why it is possible to implement pores with a hydraulic diameter of 0.05 μΐη.

If pores whose hydraulic diameter is greater than 3μηη are used, the use of the microfluidic device is a priori more delicate (for example in the choice of flow rates in the microfluidic channel 4, 40) to ensure that the However, it should be noted that the increase in this hydraulic diameter is accompanied by a decrease in the number of pores of the membrane associated with each of the openings 47, 470 covered by the membrane 5. However, this reduction in the number of pores of the membrane by opening promotes the increase of the hydraulic resistance through each opening of the microfluidic device. For this reason, it is conceivable to use pores whose hydraulic diameter goes beyond 3 μητι by limiting the conceivable range of fluid flow rates in the microfluidic channels.

In the Smith et al. Device, an opening necessarily corresponds to one and only one diffusion orifice because there is no membrane such as that proposed in the invention. As a result, the hydraulic resistance depends on the diameter of the orifice itself in the Smith et al.

The implementation of a microporous membrane 5 thus has a real advantage, since it makes it possible to dispense with a gel and the disadvantages that this gel involves.

The pore density of the microporous membrane 5 can be between 10 ³ and 10 ¹⁰ pores / cm ² . The height of the pores can be between 50 nm and 100 μητι.

Furthermore, the microporous membrane 5 can be made in various materials such as glass, quartz, silicon, silica or silicon carbide or polymers of the same nature as the polymers that can be used for the rest of the microfluidic device. It is thus possible to use polycarbonate, polyester or polyethylene terephthalate.

According to a first example, it is possible to provide a microporous membrane 5 of polycarbonate, whose pore diameter is between 0.2 μΐη and 1 μιη, for example cyclopore type from Whatman (Whatman Cyclopore ™). In a second example, one can provide a microporous membrane 5 of polyester, of which the pore diameter is between 0.4 and 3 μΐη μηη, e.g., Transwell type Corning (Corning ^® Transwell ^®). According to a third example, it is possible to provide a microporous membrane 5 of polyethylene terephthalate, the pore diameter of which is between 0.4 μηη and 8 μιτι, for example of the "Track-Etched" type from the company Becton Dickinson. These microporous membranes have the advantage of being compatible with a method of manufacturing the microfluidic device 1, which is described hereinafter with reference to FIGS. 3 (a) to 3 (d). They also have the advantage of being biocompatible and functionalizable to be specifically permeable to various molecules. Functionalisable means that the microporous membrane 5 can be chemically modified to fulfill a particular function (retention of certain species, chemical reactions, etc.).

In general, the device may have the following dimensions. The height h of the microfluidic channel may be between 1 μητι and 1,000 μητι, advantageously between 10 μΐη and 200 μηι. Its width (not shown) can be between 10 μΐη and 2 mm. The height h 'of the chamber 8 can be between 10 μΐη and 1,000 μΐη, advantageously between 50 μΐη and 200 μητι. Moreover, the distance between the entrance E and the exit S is a few centimeters.

Optical display means 18 may be associated with the microfluidic device, as mentioned above. This optical display means 18 makes it possible to know the concentration map of the stimulating molecules applied to the target cells. It also allows functional imaging of the biological response of cells to stimulation molecules. It is therefore much easier to perform correlations experimentally between the observed behavior of the target cells and the concentration map applied to them.

This observation can be made at high spatial resolution because the base of optically transparent material can be very thin. For example, one can carry out fluorescence microscopy of high resolution, or even super-resolution, with techniques such as photoactivation localization microscopy (PALM for "Photo-Activated Localized Microscopy" according to the English terminology) or stimulated emission depletion microscopy (STED for "STimulated- Emission-Depletion "according to the English terminology), using for example a base formed with a glass slide of 150 μΐη thick.

An exemplary method of manufacturing the microfluidic device 1 according to the invention is a method which comprises at least the steps of:

(a) using a stamp 1 'of an elastomeric material for printing a photocurable and / or thermosetting liquid placed on a support 2' provided with the microporous membrane 5;

(b) photo-irradiating and / or heating the liquid to form firstly, a first side wall 3 closed at its base by the microporous membrane 5 and secondly, a central wall 30 in contact with the membrane 5;

(c) sticking the lid 2 provided with orifices (not shown) on the first lateral wall 3 and on the central wall 30, on the opposite side of the support 2 'to form microfluidic channels 4, 40 in each of which a fluid can circulate ;

(d) after removing the support 2 ', sticking on the parts of the first side wall 3 and the central wall 30 made accessible by the withdrawal of the support 2', an assembly comprising at least the base 6 and said at least two second side walls 7a, 7b made of photocured and / or thermoset resin, to form the chamber 8.

This process is based on the method disclosed in WO 2008/009803.

The operation performed in step (a) is shown in Fig. 3 (a).

The stamp 1 'used during step (a) may be made of elastomeric material such as PDMS. It comprises a profile used as a mold complementary to that of the microfluidic device 1 that is to be produced. The stamp 1 'thus has a protuberance 1' has a vertical slot 1 'c to form the central wall 30 of the microfluidic device 1 that is desired. It also has a hollow zone 1 'surrounding the protrusion Va, zone in which said first side wall 3 of the microfluidic device 1 is intended to be formed. The support 2 'can also be made of PDMS and has a planar profile.

The microporous membrane 5 is previously disposed on the support 2 ', then the stamp V is pressed against the support 2'. The stamp 1 'thus wedges the membrane 5 against the support 2' via the protuberance 1'a.

Then, the volume between the stamp V and the support 2 'is filled in the appropriate quantity, especially in the hollow zone 1' b of the stamp 1 'and in the slot made in the protuberance 1'a, for example with resin photocurable and / or photopolymerizable in liquid form RL. This filling does not change the position of the microporous membrane 5, because it is wedged between the stamp 1 'and the support 2'.

The photocurable and / or photopolymerizable resin is a solution or a dispersion based on monomers and / or prepolymers. Photocure and / or photopolymerizable resins commonly used as adhesives, adhesives or surface coatings are used in the process of the invention.

Advantageously, adhesives, adhesives or surface coatings usually used in the optical field are chosen. Such resins, when irradiated and photocrosslinked and / or photopolymerized, become solid. Preferably, the solid thus formed is transparent, free of bubbles or any other irregularity.

Such resins are generally based on monomers / comonomers / pre-polymers of the epoxy, epoxysilane, acrylate, methacrylate, acrylic acid or methacrylic acid type, but there may also be mentioned thiolene, polyurethane and urethane-acrylate resins. The resins can be replaced by photocurable aqueous gels such as polyacrylamide gels and are chosen to be liquid at room temperature. The resins can also be replaced by polydimethylsiloxane (PDMS). Among the photocurable resins that can be used in the present invention, mention may be made of the products sold by Norland Optics under the trade name NOA® Norland Optical Adhesives, for example the products NOA81 and NOA60, the products marketed by the company Dymax in the range " Dymax Adhesive and light curing Systems ", the products marketed by the company Bohle in the" UV adhesive "range, the products marketed by Sartomer under the trade references SR492 and SR499.

The polymerization and / or the crosslinking of these resins is carried out by photoactivation using any appropriate means, such as irradiation with visible UV radiation, I.R.

A resin is preferably chosen which, once polymerized and / or crosslinked, is rigid and non-flexible, since the elastomeric resins tend to deform when flowing fluids under pressure in the microfluidic device 1. However, for certain applications, such as the study of the elasticity of living cells, the use of elastomeric photocurable resins is not excluded.

After filling the volume located between the stamp 1 'and the support 2' with liquid resin RL, a pressure P is then applied to the stamp 1 'to expel any excess resin. In Figure 2, the projecting parts and in particular the protuberance 1 'of stamp 1' of elastomer are in contact with the support 2 '. The liquid resin takes the form of the hollow zones of the V patch.

The structure obtained at the end of step (b) is shown in FIG. 3 (b).

During step (b), the irradiation of the resin is made in the axis perpendicular to the base of the device, through the stamp 1 '. The irradiation must be dosed so, if desired, to leave on the surface of the first side wall 3 and the central wall 30 of resins, active polymerization and / or crosslinking sites. Then, the stamp V is removed from the device. In FIG. 3 (b), the first resin side wall 3 is observed photopolymerized and / or photo-crosslinked, of complementary profile to that of the hollow zones of the stamp 1 '. In this same figure, there is also the central wall 30, which separates the microfluidic channels 4, 40.

The printing with an elastomer stamp Y in a resin in the liquid state makes it possible to obtain structures of very small sizes with a very good resolution.

In step (c), the lid 2 is then fixed with orifices for the circulation of fluid in the microfluidic channels 4, 40, on the side of said first lateral wall 3 previously in contact with the patch Y. The support 2 'can then be removed. The structure obtained at the end of step (c) is shown in FIG. 3 (c), without the openings of the lid, which are located in another plane.

The removal of the support 2 'takes place without the microporous membrane peeling off the photopolymerized resin and / or photocrosslinked, and without it being torn off or partially torn.

The cover 2 may be made of glass, silicon, a solid polymer film, a metal, a conductive or semiconductor alloy, a ceramic, quartz, sapphire, an elastomer.

Preferably, a glass slide, a polymer film or a silicon wafer is chosen. The materials used to form the lid 2 are chosen according to the application that will be made of the microfluidic device 1.

Thus, a cover 2 of optically transparent material, such as glass, is more suitable for facilitating observation, optical detection (transparency). Another asset of the glass is its very good thermal conductivity which allows a homogeneous heating of the devices.

It should be noted that the arrangement of the microporous membrane 5 at the bottom of the microfluidic channel 4 makes its use compatible with standard living cell culture protocols. Indeed, it is then conceivable that the base 6 is a lamella of glass on which a culture of living cells is carried out, this lamella then being fixed on the structure obtained at the end of step (c) to form the chamber 8 (culture chamber), as explained in the following description.

It should be noted that the manufacturing method described above can allow the manufacture of openings whose dimensions reach 5μΐη, with a pitch (distance between the respective centers of two neighboring openings) between two adjacent openings being as small as ΙΟμΐη, and therefore especially between 10μΐτι and values less than 300μ! ΎΊ, as mentioned in the article by Smith &. al. In particular, the pitch may be between 10μΐη and 250μΐη.

The assembly comprising a base 6 and two second lateral walls 7a, 7b can be made from the following process steps: (e-ι) use an open mold 3 'of elastomeric material having a support face 3'a and a cavity 3'b for receiving a photocurable and / or thermosetting liquid RL resin;

(β2) arrange the base 6 on the support face 3'a of the mold 3 ';

(e ₃ ) arrange a mask 4 'on the base 6, then photo-irradiate or heat to form said second side walls 7a, 7b.

The structure obtained at the end of steps (e-ι) to (e ₃ ) is represented in FIG. 4 (a), in the case where step (e ₃ ) consists of a photo-irradiation of the liquid resin .

The mold 3 ', such as the stamp 1' and the support 2 ', can be made of an elastomer such as PDMS.

The photocurable and / or thermosetting liquid resin used for these steps may be chosen from the possibilities already described for the liquid resin used in step (a). Preferably, the liquid resins used for steps (a) and (e-ι) to (e ₃ ) are the same. Alternatively, photocurable aqueous gels such as those previously described or polydimethylsiloxane (PDMS) could be used.

The base 6 can be chosen from the materials used for the lid. Advantageously, it will be possible to use a material optically transparent to facilitate optical viewing by a dedicated device. This optically transparent material may in particular be glass, the base 6 thus forming a glass cover usually used for the culture of living cells (CV). The use of glass also makes it possible to take advantage of existing chemical and biological surface treatments for this substrate.

The mask 4 'may have orifices 4'a, 4'b for photo-irradiating specific areas of the liquid resin to form said second sidewalls 7a, 7b of the microfluidic device.

Once the step (β3) is complete, it remains only to remove the mask 4 'and the mold 3' during a step (e) to leave only the assembly formed by said second side walls 7a, 7b and the base 6. This set is shown in Figure 4 (b).

Generally, a step (es) is then performed, the latter consisting in rinsing said assembly, for example by an ethanol / acetone mixture in 90/10 volume proportions. This rinsing makes it possible to remove all the non-photo-irradiated or unheated resin that can remain on the base 6.

Then, living cell culture (CV) is carried out before this set is disposed with the structure obtained at the end of step (c) and before starting step (d).

For this, this set must be biocompatible.

For this purpose, this assembly can be strongly photo-irradiated, for example by UV, then perform an energetic rinsing in a neutral solution, such as water for several hours.

Alternatively, it is possible to manufacture the chambers, or more generally the various elements of the device, with biocompatible materials.

Finally, a living cell culture can then be performed on the upper face 61 of the base 6, as shown in Figure 4 (c). This culture is carried out under standard conditions. In In particular, this culture can be carried out on a base 6 in the form of a conventional glass slide.

Once this culture is complete, step (d) can be performed.

The operation performed in step (d) is shown in Fig. 3 (d).

Once step (d) is performed, the microfluidic device 1 is ready for use. It comprises in particular living cells on the upper face 61 of the base 6, which is opposite to the microporous membrane 5 within the chamber 8 (culture chamber).

To operate the microfluidic device 1, it is associated, within a microfluidic system, with at least one means for supplying at least one of the microfluidic channels 4, 40 with a fluid comprising molecules capable of stimulating a target, such as living cells.

For example, two independent fluid reservoirs may be provided, one for supplying the microfluidic channel 4 with a fluid F1 comprising stimulation molecules for the target, the other for supplying the second microfluidic channel 40 with a neutral fluid F2.

An example of microfluidic channels 4, 40 that can be used with these reservoirs is shown diagrammatically in FIG. 5 (a), according to a view from above.

The fluid F1 is introduced into the microfluidic channel 4 via the inlet E ₄ , and leaves this channel 4 via the outlet S ₄ . The fluid F ₂ is introduced into the microfluidic channel 40 via the inlet E40, and leaves this channel 40 through the outlet S ₄₀ . The two microfluidic channels 4, 40 are of course separated by the central wall 30 of the microfluidic device 1.

The separation carried out by the central wall 30 recycles the fluids circulating in each of the microfluidic channels 4, 40, since no mixing can take place between these fluids. Moreover, thanks to this separation, the flow velocities of the fluids can extend over a wide range of values, for example between ΙΟΟ μΐτι / s and 10,000 μΓπ / ε or more, without risking a hydrodynamic mixing of the two fluids under the effect of shear forces. In addition, a difference in speed of circulation of the fluids between the different channels is possible without this causing any problem of operation of the device.

The fluids F ₁ , F ₂ differ only in the presence, in one of the two fluids and in low concentration, of stimulation molecules for the target cells.

It should be noted that the inputs E ₄ , E ₄ o are to be brought closer to the inlet ports 21, 22 of FIG.

FIG. 5 (b) schematizes the flow of the fluids F 1, F 2 in the various parts of the microfluidic device, which is shown schematically in a vertical sectional view.

Each fluid Fi, F ₂ is therefore intended to flow into one of the microfluidic channels 4, 40 of the microfluidic device 1, both in contact with the microporous membrane 5, but not in the chamber 8 (culture chamber). The concentration map of these molecules in the channels 4, 40 is represented by the curve C1, in steps.

The transport of the molecules (contained in the fluid Ft) capable of stimulating the living cells, between the microfluidic channel 4 and said cells installed on the base 6 of the chamber 8, is then carried out by diffusion in the chamber 8, through the microporous membrane 5.

More specifically, the transport of these molecules is carried out first by diffusion through the microporous membrane 5, then by diffusion through the chamber 8, to finally reach the upper face 61 of the base 6 of the chamber 8, face 61 on which living cells are located.

The concentration map must then stabilize in the chamber 8. In particular, at the base 6 of the chamber 8, the stabilization time t _sta b is of the order of h ' ² / D where h' is the height of the chamber 8 and D the diffusion coefficient of the molecules intended to stimulate the target cells in the chamber 8. It should be noted that to avoid stabilization too high, the height of the chamber will generally be limited to 500 μΐη.

The concentration map thus stabilized in the chamber 8 is represented by the curve C2, which is in the form of a curve representative of an "erf" type function. With this supply of microfluidic channels, it is therefore possible to obtain a very particular concentration map at the base of the chamber 8 and consequently, on the living cells that one seeks to stimulate.

The feed described above in support of Figs. 5 (a) and 5 (b) is only an illustrative example. Thus, the fluid supply of the microfluidic channels could be performed differently to obtain other types of target cell concentration maps.

Thus, according to another example, one could provide a supply means for supplying each of the two microfluidic channels 4, 40 with the fluids Fi and F ₂ , in order to obtain more complex concentration maps in the chamber 8.

The chamber 8, closed, can be replaced by a microfluidic channel comprising orifices, advantageously lateral, although no fluid is then intended to flow in this microfluidic channel when a test is in progress. However, channels can be connected to the side ports to recover secretions from living cells for the purpose of chemically analyzing them. Moreover, between two tests, it is then possible to envisage a rapid rinsing or a change of culture medium.

On the other hand, it should be noted that the target cells could also be arranged not on the basis of the chamber 8 or the microfluidic channel, but on the microporous membrane 5 itself. In such a case, the concentration map obtained at the level of the target cells corresponds to the concentration map generated in the microfluidic channels 4, 40. Indeed, in this case, the target cells are located on the side of the membrane 5, which is opposite to the side in contact with the fluids flowing in the microfluidic channels 4, 40. Moreover, it is possible to manufacture a microfluidic device 1 without a chamber or without a microfluidic channel, the target cells being placed directly on the microporous membrane 5. In this case, it is understood that the steps (e-ι) to (β3) previously described are not required for the manufacture of the device.

We will now describe another microfluidic device according to the invention in support of Figures 6 and 8 and a method of manufacturing this device in support of Figures 7 (a), 7 (b), 7 ' (a), 7 '(b) and 7 (c) to 7 (f).

The microfluidic device 101 shown in FIG. 6 comprises several microfluidic channels 401, 402 each comprising several openings 401 ', 402' on the one hand and 403 ', 404' on the other hand which open onto the microporous membrane 500. In fact, there are two microfluidic channels feeding one for 401, the six openings shown in white in Figure 8 and the other 402, the six openings shown in black in Figure 8. These openings 401 ', 402' , 403 ', 404' are arranged in the same plane P, so as to form an array of openings which is two-dimensional in this plane. The plane P is represented in FIG. 6 as well as in FIG. 8, the latter representing said openings in bottom view at this plane P.

The microporous membrane 500 extends transversely relative to the side walls of the various microfluidic channels to cover the different channels in their lower parts and thus cover the different openings.

Advantageously, the microporous membrane 500 covers all of said openings 401 ', 402', 403 ', 404' of the microfluidic channels 401, 402. This makes it possible to cover the different openings with a single membrane, which is particularly convenient when the network of openings is dense. Typically, the method according to the invention can make it possible to manufacture 5μΐτι openings, separated from one another by a no 10 μηη. The step is here defined as the distance separating the respective centers from two neighboring openings.

The microporous membrane 500 is provided with pores. The characteristics of this membrane 500 may be the same as the membrane 5 of the microfluidic device 1 described previously in support of FIGS. 2, 3 (a) to 3 (d) and 4 (a) to 4 (c).

The microfluidic device 101 also includes a lid 200 for the microfluidic channels. This cover 200 may be made of a material chosen from: glass or silicon, a non-elastomeric photocrosslinked polymer, a metal, an electrically conductive or semi-conductive alloy, a ceramic, quartz, sapphire, an elastomer.

The inlet and outlet ports for fluids for circulation in the microfluidic channels 401, 402 may be formed in this lid 200 (not shown).

The hydraulic diameter of the pores of the microporous membrane 500 prevents the fluids flowing in the microfluidic channels 401, 402 from crossing this membrane 500, only the molecules capable of stimulating the target passing therethrough. In this regard, reference should be made to the relationships (R1) to (R3) provided above and to the choice of the limiting speed below which it is considered that the membrane does not allow the fluid flowing into the microfluidic channels 401 to pass through. , 402.

The concentration map of these stimulation molecules is then generated by the choice of the fluid supply of each of the different microfluidic channels 401, 402. As can be seen in the representation of FIG. 8 (the microporous membrane 500 is not shown), the openings 401 ', 402', 403 ', 404' opening on the microporous membrane 500 form diffusion zones for the stimulation molecules, which can be likened to pixels providing chemical information to the target. The microfluidic device 101 advantageously provides a closed culture chamber 8 for said target. This chamber is thus disposed on the side of the microporous membrane 500 which is opposed to the first microfluidic channels 401, 402. The chamber has characteristics similar to those of the chamber 8 of the microfluidic device 1 described above, its dimensions however to be adapted.

Thus, the microporous membrane 500 extends transversely between the side walls of the chamber to close said chamber in its upper part.

The target may be disposed on the base 61 of the chamber 8, base 61 which is disposed on the other side of the chamber 8, with respect to the microporous membrane 500.

In a variant, it is quite possible to dispose the target in the chamber 8, directly on the microporous membrane 500, or even to dispose the target directly on the microporous membrane, in the absence of any chamber.

Again, the culture chamber 8 may be replaced by a microfluidic channel comprising orifices, advantageously lateral, no fluid however being intended to flow into this channel.

An optical display means such as the means 18 previously described and shown in FIG. 2, can also be associated with the microfluidic device 101, in particular if the base of the chamber 8 or the microfluidic channel is optically transparent.

The method of manufacturing the microfluidic device 101 is based on steps similar to the steps (a) to (d) previously described for the microfluidic device shown in Figure 2, adapting it.

Steps (a) and (b) are thus implemented to achieve the structure 200 shown in Figure 7 (b). During step (a), the stamp 10 'is thus used in an elastomeric material for printing a liquid RL photocurable and / or thermosetting placed on a support 20 'provided with the microporous membrane 500 (Fig. 7 (a)). Then, during step (b), a photo-irradiation and / or heating of the liquid is carried out to form several walls with the microporous membrane 500.

Similarly, steps (a) and (b) are implemented to provide another structure 200 'shown in Figure 7' (b). More precisely, during step (a) the stamp 10 "is used in an elastomeric material for printing a photocurable and / or thermosetting RL liquid placed on a support 20", in the absence of any microporous membrane (FIG. '(at)). Then, during step (b), photoirradiation and / or heating of the liquid is carried out to form several walls.

Then, the structures 200 ', 200 "which have been produced independently of one another are assembled to one another, by means of a new photo-irradiation or a new heating. to create a permanent bond between the liquid resins of structures 200 'and 200 "(Fig. 7 (c)).

Once the assembly is completed, one 20 "of the supports 20 ', 20" is removed to reveal a new structure 200 "formed by the assembly of the structures 200, 200'. step (c) previously described In other words, a cover 200 having orifices (not shown) is thus bonded to the various walls 3 ', 30' of the structure 200 ", on the opposite side to the microporous membrane 500 to form the microfluidic channels 401, 402.

The support 20 is then removed in contact with the microporous membrane 500 (Fig. 7 (e)). In this Figure 7 (e), the microfluidic channels 401, 402 appear. It will be noted that the microfluidic channel 401 on the one hand, and the microfluidic channel 402 on the other hand, have different depths, which makes it possible to superpose the channels in a plane, as can be seen in FIG. 9.

Then, on the membrane 500, according to step (d) described above, an assembly comprising at least the base 6 and said at least two second side walls 7a, 7b made of photocured resin and / or thermoset to form the chamber 8. This assembly is itself manufactured with a method incorporating the steps (e-ι) to (β ₃ ) described previously in support of Figures 4 (a) to 4 (c).

It should be noted that the walls of the microfluidic channels 401, 402 of the microfluidic device 101 thus obtained, as well as the walls of the chamber 8 of this device can be produced with resins as described above or, alternatively, with aqueous gels. photocurable, such as polyacrylamide gels, chosen to be liquid at room temperature. The resins can also be replaced by polydimethylsiloxane (PDMS).

To operate the microfluidic device 101 of FIG. 6, this device is associated, within a microfluidic system, with at least one means for feeding at least one of the microfluidic channels 401, 402 with a fluid comprising molecules likely to stimulate a target, such as living cells.

For example, a clean feed means (not shown) such as a fluid reservoir for each microfluidic channel 401, 402 may be provided. The connection between the reservoir and the associated microfluidic channel may be effected by means of a capillary.

The microfluidic channels 401, 402 are shown diagrammatically in FIG. 9, in a partial perspective view which represents only the channels 401, 402 supplying the openings 401 ', 402', 403 ', 404' according to the sectional view AA of FIG. Figure 8, a section that also corresponds to the view chosen to describe the manufacturing process in support of Figures 7 (a) to 7 (f), 7 '(a) and 7' (b).

The arrangement shown in Figure 9 allows to parallelize the experiments with a reduced number of connectors, insofar as a power supply will allow to feed several openings. However, the arrangement of the microfluidic channels may be different, maintaining a network of two-dimensional openings such as that shown in FIG. 8.

Thus, it can be envisaged that each channel has only one opening, so that there are as many channels as openings. Such a case is represented in FIG. 10, which represents only four channels 4010, 4020, 4030, 4040 (associated respectively with an opening 40 0 ', 4020', 4030 ', 4040') on the twelve microfluidic channels associated with the twelve FIG. 8 apertures. In this FIG. 10, each microfluidic channel may be fed with a dedicated fluid, which may in particular comprise molecules for stimulating the target.

The power supply of each channel is thus independent. Furthermore, it can then be input modulated by valves for passing at least two fluids successively in the channel.

The microfluidic device thus formed is therefore a device comparable to the device described in support of FIG. 2. However, it comprises more than two microfluidic channels.

Under these conditions, it is understood that the method of manufacturing such a device will resume the manufacturing steps described in support of Figures 3 (a) to 3 (c) and, if appropriate, the step described in Figure 3 (d), 4 (a) to 4 (c) support for forming a chamber or other microfluidic channel.

For this purpose, however, it is necessary to modify the shape of the stamp V so that the protuberance has the understanding, instead of the vertical slot the c, a hollow area in the form of a grid capable of forming the walls separating the microfluidic channels the each other, to finally obtain the network of openings of Figure 8. There is shown in Figure 11, the stamp 11 'thus modified comprising the hollow region in the form of grid' c on the protuberance with the support 2 " during the step corresponding to the step shown in Figure 3 (a).

This offers many possibilities. Indeed, it is possible to choose the microfluidic channels to supply fluid, depending on the desired map concentration of molecules that can stimulate the target.

It is also possible to feed microfluidic channels with different types of target stimulating molecules. In this way, one can carry out a precise and varied control in the space of the culture of living cells.

For example, it is possible to feed a microfluidic channel with a fluid comprising stimulation molecules and another microfluidic channel, for example immediately adjacent to the first microfluidic channel, with a fluid comprising a neutral solution. The fluids are then mixed in the chamber 8 to form a very particular concentration map at the base of the chamber, when the target cells are located on this base.

It is also possible to stimulate the target through the desired aperture 401 ', 402', 403 ', 404' with a time shift of one microfluidic channel 401, 402, 403, 404 to another. The other openings shown in Figure 8 can then be fed in the same way.

Another conceivable arrangement of the microfluidic channels is as follows.

It is indeed possible to provide only one microfluidic channel in which a fluid having stimulation molecules for the target circulates, this channel comprising several openings.

For example, it may be desired to manufacture a device comprising a channel with four openings, the microfluidic channel thus supplies fluid these four openings.

A method of manufacturing such a device is presented in support of Figs. 12 (a) to 12 (d), in the case where a chamber 8 is otherwise provided (Fig. 12 (d)).

The patch 101 'may be made of elastomeric material such as PDMS. It has a profile used as a complementary mold of that of the microfluidic device that we want to produce. The stamp 101 'thus comprises a protuberance 101' provided with several vertical slots 101 'to form the various walls 301' of the microfluidic device. It also includes a hollow zone 101 'surrounding the protuberance 101', an area in which the side wall 300 'of the microfluidic device is to be formed. The support 201 'can also be made in PDMS and has a planar profile.

The microporous membrane 500 'is previously disposed on the support 201', then the stamp 101 'is pressed against the support 201'.

Then, the volume between the stamp 101 'and the support 201' is filled in the appropriate amount, for example with photocurable and / or photopolymerizable resin in liquid form RL. After filling the volume located between the stamp 101 'and the support 201' with liquid resin RL, a pressure P is then applied to the stamp 101 'to expel any excess resin.

The structure obtained at the end of step (b) is shown in FIG. 3 (b).

The resin is irradiated through the stamp 101 '. Then, the stamp 101 'is removed from the device. In FIG. 12 (b), the walls 300 ', 301' of photopolymerized and / or photo-crosslinked resin, of profiles complementary to the hollow zones of the patch 101 'are observed.

The cover 200 'is then fixed on the side of said first lateral wall 300' previously in contact with the patch 101 '. The support 201 'can then be removed. The structure obtained at the end of this step is shown in FIG. 12 (c).

The chamber 8 is manufactured according to the method previously described in support of Figures 4 (a) to 4 (c), and then assembled with the structure shown in Figure 12 (c). This assembly operation is shown in Figure 12 (d). The flow direction of the fluid in the channel is noted F in Fig. 12 (d). It is noted that it successively feeds the various openings opening onto the membrane 500 '.

Here again, the different materials already presented above can be envisaged. The presence of the chamber 8 is not mandatory, a channel may in particular be provided in place of this chamber 8. The membrane 500 'will have the same characteristics as the membranes 5, 500 described above.

The invention thus uses a membrane whose hydraulic pore diameter is judiciously chosen to avoid the passage of the fluid from the microfluidic channels to the opposite side of the membrane, comprising for example the chamber or another microfluidic channel. The hydraulic diameter of the pores can also extend over a wide range.

The invention therefore does not require any culture medium in the form of a gel to prevent the passage of fluid from the microfluidic channels to the chamber or this other microfluidic channel.

The diffusion in the culture chamber is therefore carried out in a liquid culture medium, such as water. This diffusion in the chamber is therefore faster than in a culture medium made with a gel. This allows you to run a test more quickly and also chain different tests more quickly.

Moreover, the accuracy with which stimulation molecules reach a target obtained with the device according to the invention is excellent, and much better than with known devices.

This is notably related to the absence of gel in the chamber, which limits the multidirectional diffusion of the stimulation molecules observed with this gel.

In addition, the method according to the invention makes it possible to manufacture a network of small openings with a high surface density. Typically, the size of the openings can reach 5μηη and the distance between the respective centers of two neighboring openings can reach 10μΐη.

Finally, the sizing of the openings is totally independent of the hydraulic diameter of the pores of the microporous membrane. Thus, it is possible to manufacture a microfluidic device with small openings (size of 30 microns or less for example), associated with a microporous membrane having large pores (3 microns for example). It is also possible to manufacture a microfluidic device with large openings (size of 2000 microns for example), associated with a microporous membrane 500 having small pores (0.2 micron for example).

This leaves a great freedom of choice in the sizing of the microfluidic device, depending on the intended application.

The invention finds particular application in the field of biology, for the culture, observation and study of living cells. In particular, one can determine the chemotactic response of nerve cells to certain molecules, for example to develop neural networks. In particular, it is also possible to measure the response of cancer cells to molecules used for chemotherapy. The microfluidic system can also be used for the manufacture of biochips or for the stimulation of tissues, in particular for the production of artificial tissues.

The advantages of the invention may be of interest for other fields of application, for example to determine thresholds of toxicity of certain molecules in cosmetology.

Claims

A microfluidic system for controlling a concentration map of molecules capable of stimulating a target, for example formed by a set of living cells, characterized in that the system comprises:

a microfluidic device (1, 101) comprising:

• n _c ≥ 1 channel (s) microfluidic (s) (4, 40, 401, 402, 4010, 4020, 4030, 4040), the or each channel being provided with at least one inlet for at least one fluid and at least one outlet for this fluid;

N ₀ ≥ 2 openings (47, 470, 401 ', 402', 403 ', 404', 4010 ', 4020', 4030 ', 4040') formed in the microfluidic channel or distributed in the various microfluidic channels, said openings being arranged in the same plane so that they form a network in this plane,

the numbers n _c of microfluidic channel (s) and n ₀ of openings being connected by the relation n ₀ = Σ Π / o / c. with 1 <i < _nc and n _{0 / c} . the number of openings for the Q channel;

A chamber (8) or other microfluidic channel having a base (6) for receiving the target;

At least one microporous membrane (5, 500, 500 ') covering the network of openings, the base (6) for receiving the target being disposed on the other side of the chamber (8) or the other channel microfluidic, with respect to the microporous membrane; one or more fluid supply means (Fi, F ₂ ) for supplying the or each microfluidic channel with fluid, at least one of these fluids comprising molecules for stimulating the target;

so that when the supply means provides to the microfluidic channel or each microfluidic channel (4, 40, 401, 402, 4010, 4020, 4030, 4040) at least one of these fluids (F-ι, F ₂ ), the molecules capable of stimulating the target then diffuse, after passing through the microporous membrane (5, 500, 500 '), through the chamber (8) or said another microfluidic channel, in order to control the concentration map of molecules capable of stimulating the target in this chamber (8) or this other microfluidic channel.

2. microfluidic system according to one of the preceding claims, wherein the microporous membrane (5, 500, 500 ') is provided with pores whose hydraulic diameter is between 0.05μηι and 12μΐτι, preferably between 0.05μΐη and 3μΐη .

3. Microfluidic system according to the preceding claim, wherein the surface density of the pores of the microporous membrane (5, 500, 500 ') is between 10 ³ and 10 ¹⁰ pores / cm ² .

4. microfluidic system according to one of the preceding claims, wherein the microporous membrane (5, 500, 500 ') is made of a material selected from: glass, polycarbonate, polyester, polyethylene terephthalate, quartz, silicon, silica or silicon carbide.

5. microfluidic system according to one of the preceding claims, wherein there is provided a cover (2, 200) for the microfluidic channels, said cover being made of a material selected from: glass or silicon, a non-elastomeric photocrystalline polymer , a metal, an electrically conductive or semiconductive alloy, a ceramic, quartz, sapphire, an elastomer.

6. Microfluidic system according to the preceding claim, wherein said at least one inlet and said at least one outlet for the fluids are formed in the lid.

7. Microfluidic system according to one of the preceding claims, wherein the microfluidic channels each comprise at least one photocured and / or thermoset resin wall.

8. Microfluidic system according to the preceding claim, wherein the base (6) of the chamber (8) or the other microfluidic channel comprises is made of an optically transparent material.

9. microfluidic system according to one of claims 8 or 9, wherein the chamber or the microfluidic channel comprises side walls of photocured resin and / or thermoset.

10. microfluidic system according to one of the preceding claims, wherein there is provided a plurality of means for supplying the microfluidic channels, each of these supply means feeding one of the microfluidic channels.

11. microfluidic system according to one of the preceding claims, wherein there is provided an optical display means (18).

12. Microfluidic system according to the preceding claim, wherein the optical display means (18) implements a photoactivation localization microscopy technique or a stimulated emission depletion microscopy technique.

13. Microfluidic system according to one of the preceding claims, wherein the openings (47 ', 470', 401 ', 402', 403 ', 404') form a two-dimensional network in the plane to which they belong.

14. microfluidic system according to one of the preceding claims, wherein the respective centers of two adjacent openings are separated by a distance between 10μΐη and 250μΐη.