EP3009189A1

EP3009189A1 - Microfluid device including a flow-regulating chamber

Info

Publication number: EP3009189A1
Application number: EP14306645.4A
Authority: EP
Inventors: Jean Berthier; Théo CAMBIER; Thibault Honegger; Jérôme Larghero; Manuel Thery
Original assignee: Commissariat a lEnergie Atomique CEA; Universite Grenoble Alpes; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Current assignee: Universite Grenoble Alpes; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2014-10-16
Filing date: 2014-10-16
Publication date: 2016-04-20

Abstract

A microfluidic device (100) characterized in that it comprises at least one row (R1, R2, ..., RN) of chambers, said at least one row of chambers comprising :
· a first auxiliary channel (11) and a second auxiliary channel (12), each auxiliary channel (11, 12) having an inlet (E₁₁, E₁₂) for a fluid;
· at least two chambers (21, 22, ..., 2n) interconnecting the two auxiliary channels (11, 12), each auxiliary channel (11, 12) having for this purpose a branch connection (D₂₁, D₂₂, ..., D_2n; D'₂₁, D'₂₂, ..., D'_2n) leading to each a corresponding chamber (21, 22, ..., 2n); said first auxiliary channel (11) including a portion (P) extending between the inlet (E₁₁) of the first auxiliary channel (11) and the branch connection (D₂₁) of the first auxiliary channel (11) leasding to a first chamber (21), which portion includes means (20) for causing, during the filling of the device (100), the travel time of the fluid passing via the first auxiliary channel (11) between the inlet (E₁₁) of the first auxiliary channel (11) and a branch connection (D₂₁, D₂₂, ..., D_2n) of the first auxiliary channel (11) to be longer than or equal to the travel time of the fluid passing via the second auxiliary channel (12), a branch connection (D'₂₁, D'₂₂, ..., D'_2n) of the second auxiliary channel (12) and the corresponding chamber (21, 22,..., 2n) between the inlet (E₁₂) of the second auxiliary channel (12) and said branch connection (D₂₁, D₂₂, ..., D_2n) of the first auxiliary channel (11); and
· at least one outlet (S, S₁₂, S₂₂) for exhaust in the fluid from the auxiliary channels (11, 12), said at least one outlet being arranged after the last chamber (2n) interconnecting the auxiliary channels (11, 12).

Description

The invention relates to a microfluidic device including at least one no-flow chamber.
The term "microfluidic device" is used to mean a device having at least one dimension that is strictly less than 1 millimeter (mm).
In particular, the microfluidic device may be for receiving one or more biological cells, especially non-adhesive biological cells.
In order to study cells, they are generally housed in a chamber containing a fluid forming a culture medium for the cells.
An important aspect of this type of study is to be able to feed the cells with a culture medium that can be renewed easily, but that remains static while the study is taking place.
Specifically, a convective flow through the chamber modifies the culture conditions for the cells. Furthermore, and more critically, the presence of a convective flow can prevent tracking of cells that present little adhesion and that are moved by the flow.
Chunxiong Luo & al., "High-throughput microfluidic system for monitoring diffusion-based monolayer yeast cell structure over long time periods", Biomed Microdevices (2009), vol. 11, pp. 981-986 proposes such a microfluidic device (D1).
Figure 1(a) is a general diagram of that device 100' seen in plan view. Figures 1(b) to 1(d) are perspective views showing the various steps in filling a chamber 20' of that device 100' with a fluid that is to form a culture medium for cells.
The device 100' has a main channel 10' and a plurality of chambers 20' situated along and on either side of the main channel 10'. East chamber 20' is for receiving one or more cells for study.
Figure 1(b) is an enlargement of a diagram of Figure 1(a), showing a chamber 20' before being filled with any fluid.
When a fluid is brought in via the inlet E' of the main channel 10', the fluid enters into the chamber 20'. Nevertheless, the air that was present in the chamber 20' before it was filled with fluid cannot escape from the chamber 20'.
That is a situation that can be seen in Figure 1(c).
Also, in order to exhaust the air present in the chamber 20', the authors make use of chambers made of a material that is porous to air, e.g. polydimethyl siloxane, better known under the acronym PDMS. By placing the device under a vacuum, the pressure difference between the air present in the chamber 20' and the outside of the chamber 20', which is evacuated, together with the fact that PDMS is porous, and thus permeable to air, enables the air to be exhausted out from the chamber 20'.
After that step, a chamber 20' is thus obtained at that is filled with the fluid that is to form the culture medium for the cells.
That is the situation shown in Figure 1(d).
The chamber 20' is then not subjected to any convective flow, with the fluid passing along the main channel 10' without entering into the chamber 20'.
When it is desired to renew the culture medium, a new fluid is introduced into the device 100' and this fluid enters the chamber 20' by diffusion.
The design of that device 100' is rather constraining since it imposes making use of a porous material in order to form the chamber 20'. Without a porous material to form the chamber 20', the chamber 20' would contain air so that the device 100' could not be used.
An object of the invention is to mitigate at least one of the above-mentioned drawbacks.
In order to achieve this object, the invention provides a microfluidic device characterized in that it comprises at least one row of chambers, said at least one row of chambers comprising :

· a first auxiliary channel and a second auxiliary channel, each auxiliary channel having an inlet for a fluid;
· at least two chambers interconnecting the two auxiliary channels, each auxiliary channel having for this purpose a branch connection leading to each a corresponding chamber ;
said first auxiliary channel including a portion extending between the inlet of the first auxiliary channel and the branch connection of the first auxiliary channel leasding to a first chamber, which portion includes means for causing, during the filling of the device, the travel time of the fluid passing via the first auxiliary channel between the inlet of the first auxiliary channel and a branch connection of the first auxiliary channel to be longer than or equal to the travel time of the fluid passing via the second auxiliary channel, a branch connection of the second auxiliary channel and the corresponding chamber between the inlet of the second auxiliary channel and said branch connection of the first auxiliary channel; and
· at least one outlet for exhaust in the fluid from the auxiliary channels, said at least one outlet being arranged after the last chamber interconnecting the auxiliary channels.

The device according to the invention may also comprise the following features, taken alone or in combination:

the means for causing, during the filling of the device , the travel time of the fluid passing via the first auxiliary channel between the inlet of the first auxiliary channel and a branch connection of the first auxiliary channel to be longer than or equal to the travel time of the fluid passing via the second auxiliary channel, a branch connection of the second auxiliary channel and the corresponding chamber between the inlet of the second auxiliary channel and said branch connection of the first auxiliary channel is constituted by a means that is passive;
the passive means comprises one or more chambers;
the passive means comprise a chamber identical to at least one of the chambers interconnecting the two auxiliary channels;
each chamber interconnecting the two auxiliary channels presents a rectangular or square shape with rounded corners or a rounded shape;
a plurality of chambers interconnecting the two auxiliary channels are identical, and preferably all of the chambers interconnecting the two auxiliary channels are identical;
the chambers interconnecting the two auxiliary channels are arranged at regular intervals (d) along said auxiliary channels;
the auxiliary channels are arranged at a constant distance between at least two successive chambers of said plurality of chambers interconnecting the two auxiliary channels and preferably, between the first chamber and the last chamber interconnecting the two auxiliary channels;
the device comprises at least five chambers interconnecting the two auxiliary channels;
each auxiliary channel leads to an outlet specific to each auxiliary channel;
the auxiliary channels lead to a common outlet;
the common outlet is provided on the first auxiliary channel;
a main channel is provided having an inlet for a fluid, said main channel splitting at a main branch connection between said main channel and said auxiliary channels in such a manner that said main branch connection coincides both with the inlet of the first auxiliary channel and with the inlet of the second auxiliary channel;
there is provided a pluralty of rows of chambers, said rows being arranged in parallel with one another;
there is provided, between the inlet for the fluid and each main branch connection of the row in question, at least one main row branch connection for feeding each row with fluid from a fluid inlet common to all of the device.

Other characteristics, objects, and advantages of the invention are described in the following detailed description made with reference to the accompanying figures, in which:

· Figure 2 is a diagrammatic plan view showing an embodiment of a device in accordance with the invention;
· Figures 3(a), 3(b), 3(c) and 3(d) are likewise diagrammatic plan views showing variants of the embodiment of the invention shown in Figure 2;
· Figure 4 is a diagram showing an embodiment having a row of chambers made up of a plurality of chambers for receiving one or more cells to be studied;
· Figure 5 is a diagram showing an alternative device to the device of Figure 4;
· Figure 6 is a diagram showing an embodiment having a plurality of rows of the kind shown in Figure 4, said rows being arranged in parallel;
· Figure 7 shows a device similar to the device of Figure 6, and that has been used for performing experimental tests (24 rows of chambers arranged in parallel, 36 chambers per row);
· Figure 8 is an enlarged view of the Figure 7 device, showing one chamber;
· Figure 9 shows, as a function of time, the principle for filling the various chambers of a chamber row as shown in Figure 7, the filling being represented with the help of video pictures taken during the experiment;
· Figure 10 (a) plots up the ordinate axis the fluid flow speed inside chambers of a row of Figure 7, and along the abscissa axis the number of the chamber in question, for fluid having an inlet flow speed of 150 micrometers per second (µm/s), and Figure 10(b) plots up the ordinate axis the fluid flow speed inside chambers of the same row, and along the abscissa axis the number of the chamber in question, for fluid having an inlet flow speed of 1500 µm/s;
· Figure 11 is an experimental display showing whether fluid movement is present or absent within the various chambers of the row in question;
· Figure 12 is an enlargement of Figure 11, showing chambers in which there is no movement of fluid by convection; and
· Figure 13 shows another embodiment relating to the management of fluid inlets;
· Figure 14(a) shows a system comprising a device according to the invention and a means for feeding said device with a fluid and figure 14(b) shows another embodiment of such a system.

Figure 2 shows an embodiment of the invention.
The device 100 is a microfluidic device having a main channel 10 with an inlet E for a fluid. At a main branch connection DP, the main channel 10 splits into a first auxiliary channel 11 and a second auxiliary channel 12.
The device 100 also has a chamber 21 connecting together the two auxiliary channels 11 and 12, each auxiliary channel 11, 12 having, for this purpose, a respective branch connection D₂₁, D'₂₁ leading to the chamber 21. By "branch connection" D₂₁, D'₂₁, we can hear a connection point or junction between each auxiliary channel 11, 12 and the sub-channel SC21, SC'21 leading to the chamber 21 itself. The sub-channels will not be further referenced in the description which follows, except in figure 8 (SC2i; SC'2i).
The device 100 also has a second chamber 22 connecting together the two auxiliary channels 11 and 12, each auxiliary channel 11, 12 having, for this purpose, a respective branch connection D₂₂, D'₂₂ leading to the chamber 22.
The branch connections D₂₁, D₂₂ are located on the first auxiliary channel 11. The branch connections D'₂₁, D'₂₂ are located on the second auxiliary channel 12.
The chambers 21, 22 are advantageously identical. The auxiliary channels 11, 12 may be arranged such that the distance D between them is constant between the two chambers 21, 22.
Furthermore, the first auxiliary channel 11 includes a portion P (drawn in dashed lines) that extends between the main branch connection DP and the branch connection D₂₁ of the first auxiliary channel 11 going towards the chamber 21, which portion includes a means 20 for causing, during the filling of the device 100, the travel time of the fluid passing via the auxiliary channel 11 between the main branch connection DP and a branch connections D₂₁, D₂₂ of the first auxiliary channel 11 leading to a corresponding chamber 21, 22 to be longer than or equal to the travel time of the fluid passing via the second auxiliary channel 12, a branch connection D'₂₁, D'₂₂ and the corresponding chamber 21, 22 between the main branch connection DP and said branch connection D₂₁, D₂₂ of the first auxiliary channel 11 leading to said corresponding chamber 21, 22. The main branch connection DP coincides with the inlet E₁₁ of the first auxiliary channel 11 and with the inlet E₁₂ of the second auxiliary channel 12.
The device 100 also has an outlet S for the fluid, which outlet is common to both auxiliary channels 11 and 12. This outlet S is arranged after the last chamber 22 interconnecting the auxiliary 11, 12 channels, namely after the branch connections D₂₂ and D'₂₂. The wording "after" is with reference to the travel direction of the fluid from the inlet towards the common outlet S. This wording will have the same meaning for the remaining of the description with respect to the or each outlet.
The means described above form a row of chambers for the device.
When it is desired to fill the device 100 with a fluid, e.g. a fluid that is to form a culture medium for cells that are to be received in the chamber 21, the fluid reaches the branch connection D₂₁ via the second auxiliary channel 12, and then the chamber 21 by passing via the branch connection D'₂₁, before the same fluid reaches the branch connection D₂₁ via the first auxiliary channel 11.
As a result, the chamber 21 can be filled with fluid while expelling the air that was present therein by enabling the air to be exhausted towards the outlet S via the branch connection D₂₁.
A chamber 21 is thus obtained that does not include a pocket of air.
The same happens with the chamber 22.
A chamber 21, 22 may have a rectangular or square shape, advantageously with rounded corners, or alternatively a rounded shape. Such a rounded shape avoids any risk that air remains trapped in the chamber 21.
Unlike the teaching of document D1, air is exhausted from the chamber 21, 22 independently of the nature of the material used for making the chamber 21, 22. It is thus possible to envisage using any type of biocompatible material, and in particular materials that are inexpensive and/or that are not likely to give rise to other problems such as the culture medium evaporating or bacterial contamination through a porous wall.
Furthermore, when it is desired to renew the culture medium, the fluid propagates by diffusion in the no-flow chambers (no convective flow) more quickly, considering a similar chamber, than in the device disclosed in document D1, as for each chamber 21, 22, there are two entries, namely D₂₁, D'₂₁ for the first chamber 21 and D₂₂, D'₂₂ for the last chamber 22. Another way to see this advantage is to say that, in the frame of the invention, a bigger chamber 21, 22 may be used for a same filling time compared to document D1.
Also, once the device 100 is full, the fluid entering via the inlet E of the device 100 passes via the auxiliary channels 11 and 12 passes towards the outlet S without passing via the chamber 21, because of the pressure balance across the chamber 21, thus making it possible to obtain a chamber 21 in which there is no convective flow. Once the device 100 is full, the travel time of the fluid passing via the first auxiliary channel 11 is longer than or equal to the travel time of the fluid passing via the second auxiliary channel 12 between the entry E and the outlet S.
The presence of the chamber 22 is also of use to ensure that once the device 100 is full of fluid, this pressure balance is maintained across the chamber 21.
This can be seen more clearly below, with reference to experimental tests.
It should be observed that the means 20 for causing, during the filling, the travel time of the fluid passing via the first auxiliary channel 11 between the main branch connection DP and a branch connection D₂₁, D₂₂ to be longer than or equal to the travel time of the fluid passing via the second auxiliary channel 12, a branch connection D'₂₁, D'₂₂ and the corresponding chamber 21, 22 between the main branch connection DP and said branch connection D₂₁,D₂₂ of the first auxiliary channel 11 is advantageously constituted by a means that is passive.
As nonlimiting examples, this passive means 20 may be formed by a single chamber, a plurality chambers, or indeed one or more zigzag paths.
This passive means 20 may be arranged to define a volume that is not less than the volume of the chamber 21 connecting together the two auxiliary channels 11 and 12. In particular, the passive means 20 may be a chamber identical to the chamber 21 connecting together the two auxiliary channels 11 and 12.
A passive means 20 is advantageous because it is simpler than an active means.
However, in an alternate embodiment, the means 20 may be active, for example formed by a valve 20 controlled by a control means 201, for example an electronic control means, adapted for causing, during the filling of the device 100, the travel time of the fluid passing via the auxiliary channel 11 between the main branch connection DP and a branch connections D₂₁, D₂₂ of the first auxiliary channel 11 leading to a corresponding chamber 21, 22 to be longer than or equal to the travel time of the fluid passing via the second auxiliary channel 12, a branch connection D'₂₁, D'₂₂ and the corresponding chamber 21, 22 between the main branch connection DP and said branch connection D₂₁, D₂₂ of the first auxiliary channel 11 leading to said corresponding chamber 21, 22.
This is illustrated in figure 3(d), as an alternate embodiment to the embodiment of figure 2. Of course, an active means 20 may be envisaged for all the embodiments concerned by the invention.
In general manner, the main channel 10 may have a width lying in the range 10 µm to 500 µm. Each auxiliary channel 11 and 12 may have a width lying in the range 10 µm to 500 µm. Finally, the chamber 21 connecting together the two auxiliary channels 11 and 12 may present sides of at least 100 µm each. The depths of the channels 10, 11, and 12, and of the chamber 21 should generally be a few tens of micrometers, e.g. 20 µm. The chamber 22 may have the same dimensions as the dimensions of the first chamber 21.
The various channels 10, 11, and 12 may present a section that is circular, rectangular, or square in shape.
Finally, it should be observed that the device is to be located mainly in a plane (O; X, Y), where the reference frame (0; X, Y, Z) forms a right-handed rectangular frame of reference. The device 100 has a certain thickness (OZ axis), but the phenomena of interest (fluid passing from the inlet towards the outlet; fluid passing through each chamber 21, 22 while it is filling) take place in the two dimensions of the plane (O; X, Y). In this sense, the device constitutes a two-dimensional (2D) device. Furthermore, the device 100 is advantageously to be located in a plane (O; X, Y) that is horizontal.
Figures 3(a), 3(b), and 3(c) show variants of the embodiment of the invention shown in Figure 2, in which the fluid outlets are different.
In Figure 3(a), a respective outlet S₁₁, S₁₂ is provided for each auxiliary channel 11, 12, after the last chamber 22. In Figure 3(b), a common outlet S₁₁ is provided for both auxiliary channels 11 and 12, which outlet is provided on the first auxiliary channel 11, after the last chamber 22. In Figure 3(c), a common outlet S₁₂ is provided for both auxiliary channels 11 and 12, which outlet is provided on the second auxiliary channel 12, after the last chamber 22. Depending on the configuration, the or each outlet is more precisely located after a branch connection D₂₂ or D'₂₂. All of the other means in these variant embodiments are identical to those of the embodiment shown in Figure 2. Figure 4 shows another embodiment of the invention.
In this embodiment, it can be seen that there are a plurality of chambers 21, 22, 23, 24, ..., 2n (n > 2) connecting together the two auxiliary channels 11 and 12, and spaced apart along said auxiliary channels 11 and 12.
The chambers 21, 22, ..., 2n are advantageously identical.
The chambers 21, 22, ..., 2n are advantageously arranged at regular intervals along said auxiliary channels 11 and 12. In this situation, this means that the distance d between two successive chambers 21, 22, ..., 2n and more precisely between two successive branch connections to an auxiliary channel 11, 12 leading to one of the chambers in the plurality of chambers 21, 22, ..., 2n is constant. This is as shown in Figure 4.
In addition, the auxiliary channels 11, 12 are advantageously arranged at a constant distance D between at least two successive chambers of said plurality of chambers 21, 22, ..., 2n interconnecting the two auxiliary channels 11, 12. Preferably, the auxiliary channels 11, 12 are arranged at a constant distance D between the first chamber 21 and the last chamber 2n interconnecting the two auxiliary channels 11, 12.
From a general point of view, the device 100 may comprise at least five chambers 21, 22, ..., 2n interconnecting the two auxiliary channels 11, 12, or at least eight chambers interconnecting the two auxiliary channels 11, 12, or at least ten chambers interconnecting the two auxiliary channels 11, 12. Preferably, the device 100 will comprise at least twenty of these chambers or even at least thirthy of these chambers. Numerous chambers allow testing numerous cells in the same device.
In Figure 4, it can also be seen that there is only one common outlet S₁₁ for the fluid, which outlet is situated on the first auxiliary channel 11, after the last chamber 2n. More precisely, the outlet S₁₁ is situated after the branch connection D2n between the first auxiliary channel 11 and the last chamber 2n.
The way this outlet is arranged is merely one possible example.
It would equally be possible to provide, on the device 100 shown in Figure 4, an outlet that is common as shown in Figure 2. In a variant, it would also be possible to provide, on the device 100 shown in Figure 4, a respective outlet S₁₁, S₁₂ for each auxiliary channel 11, 12, as shown in Figure 3(a).
In a variant, it would also be possible to provide the device 100 shown in Figure 4 with a common outlet S₁₂ that is situated on the second auxiliary channel 12, after the last chamber 2n. The outlet S₁₂ would then be situated after the branch connection D'2n between the first auxiliary channel 12 and the last chamber 2n. This situation is shown in Figure 5.
An advantage of providing numerous chambers 21, 22, ..., 2n is to be able to test numerous cells in the various chambers. Furthermore, and as explained below, this also makes it possible to increase the filling rates of the device 100, while keeping certain chambers without any convective flow.
Figure 6 shows another embodiment of the invention.
In this embodiment, there are provided a plurality of rows R1, R2, R3 of chambers, each row having a plurality of chambers (i.e. n = 2 or n > 2) interconnecting two auxiliary channels. In Figure 6, it can also be seen that the row R2 corresponds to the diagram of Figure 4 with n = 8 (the first chamber 20 forming the means for providing the delay).
The rows R1 and R3 are arranged in parallel with the row R2.
The rows R1 and R3 are advantageously identical to the row R2.
In order to feed these rows, it is then appropriate to provide the main channel 10 with a main row branch connection DPR suitable for feeding each of the rows R1, R2, and R3. This branch connection DPR is upstream from the main branch connection DP, "upstream" being relative to the flow direction of the fluid in the device 100. This branch connection DPR serves to feed the main channel 102, 103 of each additional row R1, R3. Once again, the main branch connection DP coincides with the inlet E₁₁ of the first auxiliary channel 11 and with the inlet E₁₂ of the second auxiliary channel 12.
Furthermore, as shown in Figure 6, since the rows R1, R2, and R3 are identical, each has a single respective outlet S_11,R1, S_11,R2, or S_11,R3 arranged as shown in the diagram of Figure 4.
Nevertheless, it is equally possible to provide, one outlet the auxiliary channel for each row R1, R2, R3, as shown in the diagram of Figure 3(a), or one outlet in common as shown in the diagram of Figure 2, i.e. a single common outlet connected to all of the auxiliary channels of each row. In a variant, a common outlet may be provided connected to one of the auxiliary channels of each row, which may be an auxiliary channel of the same type as the channel 11 (Figure 4), or which may be an auxiliary channel of the same type as the channel 12 (Figure 5).
Naturally, Figure 6 merely shows one example, and more generally it is possible to provide N rows, with N > 1.
In Figure 7, there can be seen a device 100 in accordance with the invention that has been used for performing experimental tests. This experimental device has 24 rows (N = 24; the rows R1 and R24 being identified in Figure 7) and 36 chambers the row ( chamber numbers 1 and 36 are identified in Figure 7).
All of the rows are identical.
All of the chambers of each row are identical. In particular, this means that, specifically, the means 20 for providing a time offset is a chamber identical to the other chambers.
It should be observed that, for each row, the chamber numbered 1 corresponds to a chamber 20 (cf. Figures 2 to 6) forming the passive means for imparting a time offset while filling of the experimental device with the fluid. Thus, for each row, the chamber numbered 2 corresponds to the chamber 21 (cf. Figures 2 to 6) and the chamber numbered 36 corresponds to the chamber 2n with n = 35 (cf. Figures 4 to 6).
In this experimental device, each row R1 to R24 provides an outlet arranged in accordance with Figures 4 and 6.
A characteristic length L of this experimental device is shown in Figure 7; namely L = 12.5 mm
Figure 8 shows more precisely the characteristic widths of the experimental device in a chamber 2i (0 ≤ i ≤ n=35).
The first auxiliary channel 11 presents a section that is rectangular, of width (axis OY) equal to 75 µm. The chamber 2i presents a shape that is substantially square with rounded corners, and each side presents a length of 300 µm (axis OY or axis OX). The second auxiliary channel 12 presents a section that is rectangular, of width (axis OY) equal to 100 µm. The passage connecting the chamber 2i to each of the auxiliary channels 11, 12 presents a width of 50 µm. For all of these means, the depth (axis OZ) is 20 µm.
Furthermore, at the inlet E, the channel presents a width of 500 µm, still with a depth of 20 µm.
Specifically, the experimental device was made of PDMS.
More precisely, the experimental device was made as follows.
A layer of positive photosensitive resist (AZ9260; AZ Electronic Materials) was spin-coated onto a silicon substrate at 750 revolutions per minute (rpm) for 60 seconds (s). That produced a resist layer having a thickness of 20 µm.
The resist, once on its substrate, was pre-baked for 1 minute (min) at 60°C and then for 4 min at 110°C.
It was exposed to ultraviolet (UV) through a photomask (Toppan) laying bare the areas of interest (for obtaining the shape desired for the experimental device) for 50 s (exposure of 35 milliwatts per square millimeter (mW/mm²) at a wavelength of 405 nm, and of 16 mW/mm² at a wavelength of 365 nm; those being the two wavelengths of the lamp used) onto a mask aligner (MJB4, SussMicroTec), developed in AZ400K 1:4 (AZ Electronic Materials) for 60 s.
It was washed in deionized water.
The layer of photosensitive resist was then dried by air-blow.
PDMS (kit sylgard, Dow Corning) was mixed with a curing agent, degassed, poured into a mold, and cured for 1 hour (h) at 100°C on a hot plate.
The layer of PDMS was then removed.
The PDMS having the shape desired for making the experimental device, and a standard glass slide (borosilicate glass) were oxidized under plasma for 10 s at 100 W (Femto, Diener Electronics) and put into contact to ensure bonding and prevent any leakage of fluid in use. Prior to bonding, orifices were formed in the PDMS for subsequent connection with the fluid feed.
Thereafter, the connections were made for the fluid feeds. For this purpose, the device as obtained at this stage was connected to a pump via capillaries, the capillaries being inserted in the orifices in the PDMS. The other ends of the capillaries were connected to a reservoir for containing the fluid for feeding to the experimental device.
The fluid used was water.
Two tests were performed, one with an inlet flow speed E of V_E = 150 µm/s, and the other with an inlet flow speed of V_E = 1500 µm/s.
The results obtained are described below with reference to Figures 10(a), 10(b), 11, and 12.
Nevertheless, to begin with, Figure 9 is an experimental display showing how the experimental device becomes filled as a function of time.
At time t = 0.6 s it can be seen that the fluid has filled the chamber 21 up to the branch connection D₂₁ by passing via the second auxiliary channel 12, before the fluid reaches the same branch connection D₂₁ by traveling along the first auxiliary channel 11.
It can be seen that this same principle applies for the other chambers at multiples of 0.6 s.
Figure 10(a) is an experimental result obtained for fluid admitted into the experimental device at a flow speed of V_E = 150 µm/s, after fluid has filled the entire experimental device.
The chamber number for the row in question is plotted along the abscissa axis ("position"), and the fluid flow speed V_C through the chamber in question is plotted up the ordinate axis (it should be recalled that the chamber numbered 1 is the chamber that is used for imparting the time offset; it corresponds to the chamber 20 in Figures 4 to 6).
It should be observed that the chambers numbered 2 and 3 are subjected to a convective flow, i.e. fluid passes through these chambers by convection.
In contrast, the chambers numbered 4 to 32 are without flow. This is associated with the fact that pressure is balanced, on either side of each chamber in question.
The last chambers, i.e. the chambers numbered 33 to 36, are likewise subjected to the passage of fluid. The inventors believe that this is associated with the fact that the second auxiliary channel does not continue beyond the last chamber 2n, specifically on the second auxiliary channel 12, which implies a special limit condition.
In Figure 10(b), the inlet flow speed is multiplied by a factor of 10 and is thus given by V_E = 1500 µm/s.
Qualitatively speaking, the results shown in Figure 10(b) are similar to those shown in Figure 10(a).
In contrast, qualitatively speaking, there are significant differences.
Specifically, on comparing the results of Figure 10(b) with the results of Figure 10 (a), it can be seen that the absolute values for fluid flow speed within a given chamber are higher. This is easily understood since the inlet fluid flow speed is likewise higher (by a factor of 10).
Nevertheless, an interesting aspect lies in the fact that, for an inlet fluid flow speed of 150 µm/s, only the chambers numbered 2 and 3 are subjected to passing a (convective) flow, whereas for the flow speed of 1500 µm/s, it is the chambers numbered 2, 3, 4, and 5 that are subjected to a flow. Furthermore, when considering a given chamber, e.g. the chamber numbered 2, it can be seen that the ratio of the fluid flow speed through the chamber divided by the fluid flow speed at the inlet to the device is smaller for an inlet fluid flow speed of 150 µm/s (ratio = 4/150) than it is for an inlet fluid flow speed of 1500 µm/s (ratio ≅ 50/1500; a reduction of about 20%).
Reducing the fluid flow speed at the inlet thus makes it possible to limit the number of chambers that are subjected to a convective flow of fluid.
This shows clearly that, for an inlet flow speed to the device that is small enough, it is entirely possible to obtain a chamber in which there is no convective flow with a device having only two chambers numbered 2 and 3 (i.e. a chamber 21 and a chamber 22 as shown in Figures 2 and 3; with the chamber 20 serving to provide the time offset not being taken into consideration and with the chamber 21 having no flow). Under such circumstances, the inventors consider that the chamber 22 plays a role in balancing pressures on either side of the chamber 21. The chamber 21 can thus be entirely without convective flow, under certain flow speed conditions, when using a device 100 as shown in any one of the devices shown in Figures 2, 3(a), 3(b), or 3(c).
In practice, it is nevertheless advantageous to provide for a plurality of chambers, with n >> 2 in order to study numerous cells at the same time and in order to be able to change the culture medium quickly, typically in a few minutes. Typically, it is possible to provide more than ten chambers 21, 22, ..., 2n per row, and more particularly several tens of chambers of this type.
Figure 11 shows a display obtained during testing with an inlet fluid flow speed V_E = 1500 µm/s. It confirms (to the left and to the right) that certain chambers are subjected to a convective flow, but above all that other chambers (in the middle) are not subjected to any convective flow while fluid is flowing in the auxiliary channels 11 and 12. For this purpose, the fluid contained particles identifiable with the method used for producing the display (the particles appear white).
Figure 12 is an enlargement of Figure 11 showing chambers without flow.
Another interesting aspect of the invention relates to renewing the fluid.
In this respect, an additional test was performed in which the colorless fluid (water with particles) was changed for a colored fluid. Those tests reveal that changing the fluid lead to no change concerning the absence of convective flow in the "no-flow" chambers, but that the colored fluid did penetrate into those chambers by diffusion. With the device of the invention, it is thus easy to change the culture medium for the cells, without modifying flow conditions.
It should be noted that, when it is desired to introduce biological cells in the device, it is advantageously carried out by including such cells in the fluid during the filling of the device.
In addition, the device 100 may be made of other types of materials than PDMS, as a porous medium is not necessary for the chambers 20, 21, ..., 2n.
For example, the device 100 may be made of plastics such as polystyrene (PS), polycarbonate (PC), polymethylmethacrylate (PMMA) or polypropylene (PP). The companies Greiner or Ibidi provides such plastics for microfluidic applications.
Indeed, using a porous material is not ideal since such a material can give rise to problems such as the culture medium evaporating or bacterial contamination.
According to other examples, the device 100 may be made of Thermoset Polyester (TPE), Polyurethane Methacrylate (PUMA) or NorlandAdhesive 81 (NOA81).
Finally, it should be observed that for all of the above-described embodiments, only one fluid inlet E is provided.
This is particularly advantageous.
Nevertheless, in all of the above-described embodiments, it is entirely possible to envisage providing a plurality of inlets for feeding the various auxiliary channels 11, 12 with fluid.
In particular, it is possible to envisage providing one inlet for each auxiliary channel 11, 12. Figure 13 is a diagram showing this possibility, based on modifying the embodiment shown in Figure 2. Under such circumstances, the first auxiliary channel 11 includes a portion P that extends between the inlet E₁₁ of this first auxiliary channel 11 and the branch connection D₂₁ of the first auxiliary channel 11 going towards a first chamber 21, which portion includes a means 20 for causing, during the filing of the device, the travel time of the fluid passing via the first auxiliary channel 11 between the inlet E₁₁ of the first auxiliary channel 11 and a branch connection D₂₁, D₂₂ of the first auxiliary channel 11 to be longer than or equal to the travel time of the fluid passing via the second auxiliary channel 12, a branch connection D'₂₁, D'₂₂ of the second auxiliary channel 12 and the corresponding chamber 21, 22 between the inlet E₁₂ of the second auxiliary channel 12 and said branch connection D₂₁, D₂₂ of the first auxiliary channel 11.
Naturally, it is possible to envisage one inlet per auxiliary channel for all of the embodiments described with reference to Figures 3(a), 3(b), 3(c), 4, 5, and 6.
Whatever the embodiment considered, the invention also proposes a system 1000 comprising:

a device 100 according to the invention;
a means 101 for feeding said device 100 with a fluid.

This is illustrated in figure 14(a), with, in this example, a device 100 having one entry E and one outlet S.
For example, the means 101 may be a reservoir. In that case, the reservoir 101 will contain the fluid. For the application to the study of cells, this reservoir will be a culture medium.
A fluidic connection 105 may be envisaged between the means 101 and the entry E of the device 100 according to the invention.
Advantageously, the system 1000 will comprise a means 102 for receiving the fluid exhausted from said device 100, as illustrated in figure 14(a). A fluidic connection 106 may be envisaged between the outlet S of the device 100 according to the invention and said means 102. For example, the means 102 may be reservoir, as illustrated in figure 14(a).
In some cases, the system 1000 may further comprise a fluid connection 103 linking said means 102 to said means 101 and comprising a pump 104 leading the fluid from said means 103 to said means 101.
This is illustrated in figure 14(b).

Claims

A microfluidic device (100) characterized in that it comprises at least one row (R1, R2, ..., RN) of chambers, said at least one row of chambers comprising :
· a first auxiliary channel (11) and a second auxiliary channel (12), each auxiliary channel (11, 12) having an inlet (E₁₁, E₁₂) for a fluid;

· at least two chambers (21, 22, ..., 2n) interconnecting the two auxiliary channels (11, 12), each auxiliary channel (11, 12) having for this purpose a branch connection (D₂₁, D₂₂, ..., D_2n; D'₂₁, D'₂₂, ..., D'_2n) leading to each a corresponding chamber (21, 22, ..., 2n); said first auxiliary channel (11) including a portion (P) extending between the inlet (E₁₁) of the first auxiliary channel (11) and the branch connection (D₂₁) of the first auxiliary channel (11) leasding to a first chamber (21), which portion includes means (20) for causing, during the filling of the device (100), the travel time of the fluid passing via the first auxiliary channel (11) between the inlet (E₁₁) of the first auxiliary channel (11) and a branch connection (D₂₁, D₂₂, ..., D_2n) of the first auxiliary channel (11) to be longer than or equal to the travel time of the fluid passing via the second auxiliary channel (12), a branch connection (D'₂₁, D'₂₂, ..., D'_2n) of the second auxiliary channel (12) and the corresponding chamber (21, 22,..., 2n) between the inlet (E₁₂) of the second auxiliary channel (12) and said branch connection (D₂₁, D₂₂, ..., D_2n) of the first auxiliary channel (11); and

· at least one outlet (S, S₁₂, S₂₂) for exhaust in the fluid from the auxiliary channels (11, 12), said at least one outlet being arranged after the last chamber (2n) interconnecting the auxiliary channels (11, 12).
A device (100) according to claim 1, wherein the means (20) for causing, during the filling of the device (100), the travel time of the fluid passing via the first auxiliary channel (11) between the inlet (E₁₁) of the first auxiliary channel (11) and a branch connection (D₂₁, D₂₂, ..., D_2n) of the first auxiliary channel (11) to be longer than or equal to the travel time of the fluid passing via the second auxiliary channel (12), a branch connection (D'₂₁, D'₂₂, ..., D'_2n) of the second auxiliary channel (12) and the corresponding chamber (21, 22,..., 2n) between the inlet (E₁₂) of the second auxiliary channel (12) and said branch connection (D₂₁, D₂₂, ..., D_2n) of the first auxiliary channel (11) is constituted by a means that is passive.
A microfluidic device (100) according to the preceding claim, wherein the passive means (20) comprise one or more chambers.
A microfluidic device (100) according to claim 2 or claim 3, wherein the passive means (20) comprises a chamber identical to at least one of the chambers (21, 22, ..., 2n) interconnecting the two auxiliary channels (11, 12).
A microfluidic device (100) according to any preceding claim, wherein each chamber (21, 22, ..., 2n) interconnecting the two auxiliary channels (11, 12) presents a rectangular or square shape with rounded corners or, a rounded shape.
A microfluidic device (100) according to any preceding claim, wherein a plurality of chambers (21, 22, ..., 2n) interconnecting the two auxiliary channels (11, 12) are identical, and preferably all of the chambers (21, 22, ..., 2n) interconnecting the two auxiliary channels (11, 12) are identical.
A microfluidic device (100) according to any preceding claim, wherein the chambers (21, 22, ..., 2n) interconnecting the two auxiliary channels (11, 12) are arranged at regular intervals (d) along said auxiliary channels (11, 12).
A microfluidic device (100) according to any preceding claim, wherein the auxiliary channels (11, 12) are arranged at a constant distance (D) between at least two successive chambers of said plurality of chambers (21, 22, ..., 2n) interconnecting the two auxiliary channels (11, 12) and preferably, between the first chamber (21) and the last chamber (2n) interconnecting the two auxiliary channels (11, 12).
A microfluidic device (10) according to any preceding claim, comprising at least five chambers interconnecting the two auxiliary channels (11, 12).
A microfluidic device (100) according to any preceding claim, wherein each auxiliary channel (11, 12) leads to an outlet (S₁₁, S₁₂) specific to each auxiliary channel.
A microfluidic device (100) according to any one of claims 1 to 9, wherein the auxiliary channels (11, 12) lead to a common outlet (S, S₁₁, S₁₂).
A microfluidic device (100) according to the preceding claim, wherein the common outlet (S₁₁) is provided on the first auxiliary channel (11).
A microfluidic device (100) according to any preceding claim, wherein a main channel (10) is provided having an inlet (E) for a fluid, said main channel (10) splitting at a main branch connection (DP) between said main channel (10) and said auxiliary channels (11, 12) in such a manner that said main branch connection (DP) coincides both with the inlet (E₁₁) of the first auxiliary channel (11) and with the inlet (E₁₂) of the second auxiliary channel (12).
A microfluidic device (100) according to any preceding claim, wherein there is provided a pluralty of rows (R1, R2, ..., RN) of chambers, said rows being arranged in parallel with one another.
A microfluidic device (100) according to the preceding claim, wherein there is provided, between the inlet (E) for the fluid and each main branch connection (DP) of the row in question (R1, R2, R3), at least one main row branch connection (DPR, DPR1, DPR2, DPR3) for feeding each row (R1, R2, R3) with fluid from a fluid inlet (E) common to all of the device.