Device and method for capturing cell pairs and method for analyzing cell pairs in real time

Abstract

The invention relates to a device for capturing at least one pair of cells in a solution containing at least one first cell (C1) of a first type and at least one second cell (C2) of a second type, said device comprising:-a microfluidic channel (3) adapted for a unidirectional flow (F) of the solution; -a first trap (1) comprising a pair of first fingers (10a, 10b) arranged in the microfluidic channel (3), at least one of the first fingers (10a, 10b) being coupled with a respective first actuator (11a, 11b) configured to adjust the first trap (1) in a direction transverse to the flow (F) between an open position allowing the first cells to flow between the first fingers (10a, 10b) and a closed position allowing the first cells to flow between the first fingers (10a, 10b); a closed position adapted to the size of the first cell to allow capture of the first cell between the first fingers (10a, 10b); 15-a second trap (2) comprising a pair of second fingers (20a, 20b) arranged in the microfluidic channel (3), at least one of the second fingers (20a, 20b) being coupled with a respective second actuator (21a, 21b) configured to adjust the second trap (2) in a direction transverse to the flow (F) between an open position allowing second cells to flow between the second fingers (20a, 20b) and a closed position allowing second cells to flow between the second fingers (20a, 20b). A closed position adapted to the size of the second cell to allow capture of the second cell between the second fingers (20a, 20b); wherein the first trap (1) is arranged relative to the second trap (2), when the first trap and the second trap are in a closed position, so as to form a cell pair comprising captured first and second cells, such that the second cells physically or chemically interact with the first cells.

Description

Background Art

[0001] Cell-cell interactions play a vital role in various biological systems, especially in immunity, where cell pairing initiates and mediates many key immune responses (selection, proliferation, differentiation) and functional immune responses (cell lysis, cytokine and antibody production) (RD Schreiber, LJ Old, and MJ Smyth, Science, Vol. 331, No. 6024, pp. 1565-70, 2011). In this context, a better understanding of the interaction dynamics between immune cells and their cell partners is fundamental.

[0002] These interactions are usually studied by activating cells in batch co-culture, mixing cell populations and initiating contact by brief centrifugal co-precipitation, and then piecing together the measurements of independent analyses performed at different time points. Although this batch co-culture reveals key information about these interactions, unfortunately, the results are averaged between many different cell types and interaction combinations (B. Dura and J. Voldman, Current opinion in immunology, Vol. 35, pp. 23-9, 2015). These technical methods actually mask the intrinsic cellular heterogeneity in terms of interaction potential, variation in antigen presenting cells and duration of contact, all of which are known to modulate immune responses (B. Dura and J. Voldman, Current opinion in immunology, Vol. 35, pp. 23-9, 2015). In tumor immunology, there is increasing evidence that population-wide measurements that mask the behavior of individual cells cannot reflect the fate of the tumor.

[0003] Furthermore, while immunotherapy using immune checkpoint inhibitors (ICIs) has completely changed the therapeutic landscape for cancer treatment (A. Ribas and JD Wolchok, Science, Vol. 359, No. 6382, pp. 1350-1355, 2018), not all types of cancer respond equally well to ICIs, and even among responsive cancers, only a subset of patients experience durable responses and good long-term outcomes. Heterogeneity in immune responses within and between patients can explain the variability in ICI efficiency. Therefore, it is critical to establish methods for real-time analysis of single cell interactions and behaviors in a non-artificial cellular environment to identify reliable predictive biomarkers to distinguish ICI responders from non-responders and to identify candidates for rational combination therapies, personalized approaches that are now considered fundamental to the field of tumor immunology.

[0004] There are several micro tools for studying the interaction of single cells. A common method is to separate discrete numbers of cells in microwells, microchambers or droplets and monitor their interactions using multiple measurements (B. Dura and J. Voldman, Current opinion in immunology, Vol. 35, pp. 23-9, 2015). Although these methods make it possible to resolve the relationship between different immune responses (B. Dura and J. Voldman, Current opinion in immunology, Vol. 35, pp. 23-9, 2015), they still have some limitations because they cannot study early signal transduction dynamics (calcium ion entry, immune synapse formation...), nor can they study its correlation with subsequent functional cellular events.

[0005] In order to analyze or modify the immune status of a patient, it is also very important to be able to isolate individual living cells (e.g., individual T lymphocytes or cancer cells) after their interaction. In fact, postsynaptic cell isolation is very important for analyzing single cell omics, or for expanding single cells in vitro, analyzing single cells or adoptive transfer of tissues for in vivo studies.

[0006] Microfluidics is well suited to studying single cell behavior and isolating live postsynaptic cells individually. Some microfluidic devices even enable the study of cell interactions:

[0007] At the 24th International Conference on Miniaturized Systems for Chemistry and Life Science on October 4-9, 2020, Fairuk A. Shaik et al. described a device for pairing cells of different sizes in Pairing cells with different dimensions in a microfluidic device using a unidirectional flow. The device includes a microfluidic channel in which an array of capture sites is arranged. Each capture site consists of a three-layer structure, including, from the bottom to the top of the microfluidic channel, a first layer that allows a solution containing cells to flow along the microchannel, a second layer that forms a trap for smaller cells of the expected size, and a third layer that forms a trap for larger cells of the expected size.

[0008] Another device for pairing cells is described in Shaik Faruk Azam et al., Pairing cells of different sizes in a microfluidic device for immunological synapse monitoring. Lab Chip. 2022; 22(5): 908-20. The device comprises two parallel microfluidic channels separated by a wall presenting a synaptic opening, and micropillars are arranged on both sides of the synaptic opening to form corresponding capture sites adapted to retain cells in a solution flowing in the corresponding channel.

[0009] However, in these devices, the capture sites have a predetermined fixed size and cannot effectively pair cells with sizes different from the expected size. In addition, once cells are captured in the capture sites, it is usually necessary to reverse the flow in the microfluidic channel to release the cells from the capture sites, which is inconvenient.

[0010] Therefore, despite the existence of very complex systems, there is currently no microfluidic device that is able to: i) analyze the naturally occurring physical interactions and behaviors of primary cells in a non-artificial environment, ii) immobilize large numbers of cell pairs without affecting their viability, iii) separate cells after cell interaction to efficiently expand cells in vitro, where the cells are preferably non-frequent cells of different sizes (e.g., stem cells, dormant cells, etc.).

[0011] More precisely, there is currently no such microfluidic cell pairing device that enables the study of cell-cell interactions between live primary human lymphocytes and their partners (e.g., primary cancer cells) and can (i) screen therapeutics or candidate molecules and (ii) retrieve individual T cells or cancer cells after synapse formation. More precisely, there is no solution to decipher the molecular events occurring at the scale of the immune synapse between primary cancer cells and key T cells in patients, either in a clinical setting or in a tumor dormant setting. Summary of the invention

[0012] The present invention provides a device for capturing at least one cell pair in a solution, wherein the solution contains at least one first cell of a first type and at least one second cell of a second type, the device comprising:

[0013] -Microfluidic channels adapted for unidirectional flow of solutions;

[0014] - a first trap comprising a pair of first fingers arranged in the microfluidic channel, at least one of the first fingers being coupled to a corresponding first actuator, the first actuator being configured to adjust the first trap in a direction transverse to the flow between an open position allowing the passage of a first cell between the first fingers and a closed position adapted to the size of the first cell to allow the capture of the first cell between the first fingers;

[0015] - a second trap comprising a pair of second fingers arranged in the microfluidic channel, at least one of the second fingers being coupled to a respective second actuator, the second actuator being configured to adjust the second trap in a direction transverse to the flow between an open position allowing the second cell to pass between the second fingers and a closed position adapted to the size of the second cell to allow capture of the second cell between the second fingers;

[0016] Wherein the first trap is arranged relative to the second trap so that when the first trap and the second trap are in a closed position, a cell pair is formed comprising a captured first cell and a second cell such that the second cell physically or chemically interacts with the first cell.

[0017] In some embodiments, in the closed position, the second trap is larger than the first trap so as to allow a second cell that is larger than the first cell to be captured in the second trap.

[0018] In some embodiments, each first finger is coupled to a corresponding first actuator, and each second finger is coupled to a corresponding second actuator.

[0019] The first and second actuators may be configured to move the first trap relative to the second trap to separate the first cell from the second cell and to open the first and second traps to release the first and second trapped cells in the flow.

[0020] In some embodiments, each first finger or second finger is coupled to a respective first actuator or second actuator through a respective first rod or second rod that is substantially perpendicular to the flow.

[0021] Each of the first and second rods may support at least two first fingers or second fingers, respectively, to form at least two first traps and second traps that are simultaneously adjustable by the first and second actuators, respectively.

[0022] In some embodiments, the device may also include two pairs of foldable beams extending along the flow and connected to one of the first rod and the second rod, each end of the foldable beam is fixed relative to the microfluidic channel, wherein each foldable beam can be deformed by a corresponding first actuator or a second actuator to move the corresponding first rod or the second rod in a direction transverse to the flow, thereby adjusting the first trap or the second trap.

[0023] In some embodiments, the device also includes a control unit configured to receive sizes of the first cell and the second cell, and to control the first actuator and the second actuator respectively based on the received sizes of the first cell and the second cell to adjust the distance between the first fingers and the distance between the second fingers in the closed position of the first trap and the second trap.

[0024] The apparatus may further include a measuring unit adapted to measure the sizes of the first cell and the second cell in real time, and the control unit is configured to receive the measured sizes of the first cell and the second cell in real time.

[0025] In some embodiments, the device further comprises at least one mechanical sensor or electrical sensor adapted to detect cells that have been trapped in the respective first trap, the second trap, and, if applicable, the third trap.

[0026] In some embodiments, at least one first actuator is configured to adjust the size of the first trap in a closed position after capturing a first cell to allow a third cell to be captured in the first trap to form a cell triplet with the second cell captured in the second trap, so that the first cell, the second cell, and the third cell physically or chemically interact.

[0027] In some embodiments, the device further comprises a third trap comprising a pair of third fingers arranged in the microfluidic channel, at least one of the third fingers being coupled to a corresponding third actuator, the third actuator being configured to adjust the third trap in a direction transverse to the flow between an open position allowing a third cell to pass between the third fingers and a closed position adapted to a size of the third cell to allow capture of the third cell between the third fingers;

[0028] Wherein, the third trap is arranged relative to the first trap and the second trap so that when the first trap, the second trap and the third trap are in a closed position, a cell triplet containing the captured first cell, the second cell and the third cell is formed, so that the first cell, the second cell and the third cell undergo physical or chemical interaction.

[0029] In some embodiments, the device further comprises an array of electrically isolated electrodes arranged at the bottom of the microfluidic channel such that an overlapping region of two electrodes is located below each trap, at least one surface of each electrode is exposed in a groove in the overlapping region, and each electrode is connected to a power source so as to selectively apply a potential difference to the solution in each overlapping region.

[0030] Another object of the present invention is a method for capturing at least one cell pair in a solution containing at least one first cell of a first type and at least one second cell of a second type, the method comprising:

[0031] - causing the solution to flow in a microfluidic channel of a device as described above;

[0032] - activating at least one first actuator to close the first trap, the size of said first trap in the closed position being adapted to the size of the first cell;

[0033] - capturing a first cell in a first trap;

[0034] - activating at least one second actuator to close the second trap, the dimensions of the second trap in the closed position being adapted to the dimensions of the second cell;

[0035] - capturing a second cell in a second trap, the second cell forming a cell pair with the first cell, the second cell interacting physically or chemically with the first cell.

[0036] Another object of the invention is a method for capturing cell triplets in a solution containing at least one first cell of a first type, at least one second cell of a second type different from the first type, and at least one third cell of a third type, the method comprising:

[0037] - causing the solution to flow in a microfluidic channel of a device as described above;

[0038] - activating at least one first actuator to close the first trap, the size of said first trap in the closed position being adapted to the size of the first cell;

[0039] - capturing a first cell in a first trap;

[0040] - activating at least one first actuator to adjust the size of the first trap in the closed position to adapt to the size of the first cell and the third cell;

[0041] - Capture a third cell in the first trap;

[0042] - activating at least one second actuator to close the second trap, the dimensions of the second trap in the closed position being adapted to the dimensions of the second cell;

[0043] - capturing a second cell in a second trap, wherein the second cell forms a triplet with the first cell and the third cell, and the first cell, the second cell and the third cell interact physically or chemically.

[0044] In some embodiments, the first trap and the second trap are in an initial open position, wherein the first trap is away from the second trap in a direction transverse to the flow, and after the first cell and the second cell are captured in the first trap and the second trap, the first actuator and / or the second actuator is activated to bring the first trap and the second trap closer to each other to produce cell pairs or cell triplets.

[0045] The method may further include releasing at least one of the first cell, the second cell, and, if applicable, the third cell by activating the first actuator and / or the second actuator to move at least one of the first trap and the second trap so as to move away from the other trap in a direction transverse to the flow, and opening at least one of the first trap and the second trap to release the cell captured in the corresponding trap.

[0046] Alternatively, the method may include releasing at least one of the first cell, the second cell, and (if applicable) the third cell from a selected trap by applying a potential difference to overlapping electrodes below the trap that is greater than the potential difference that triggers electrolysis, dielectrophoresis, or electroosmosis of a solution in the corresponding recess so as to generate a bubble adapted to push the first cell, the second cell, and / or the third cell out of the trap.

[0047] Another object of the present invention is a method for analyzing real-time interactions of at least one pair of cells or a trio of cells, comprising:

[0048] - capturing at least one cell pair of a cell triplet using a method as described above;

[0049] - using at least one of an optical sensor, an electrical sensor, a mechanical sensor and a chemical sensor, acquiring data related to the interaction of the cell pairs or cell triplets.

[0050] The method may further comprise exposing at least one captured cell pair or cell triplet to one or more solutions having a defined pH value and / or a defined viscosity, the pH value or viscosity being selected to mimic cell interactions under defined circumstances.

[0051] The present invention also provides a system for analyzing real-time interactions of at least one pair of cells or triplet of cells, comprising:

[0052] - a device as described above, and

[0053] - At least one of an optical sensor, an electrical sensor, a mechanical sensor, and a chemical sensor configured to acquire data related to the interaction of a pair or a triplet of cells captured by the device. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Other features and advantages of the present invention will become apparent from the following detailed description based on the accompanying drawings, in which:

[0055] - Figures 1A to 1E shows the sequence of operations of a device according to an embodiment of the present invention allowing the capture of cell pairs;

[0056] - FIG. 2A to FIG. 2F shows the sequence of operations of a device according to an embodiment of the invention allowing the capture of cell triplets;

[0057] - FIG. 3A to FIG. 3C shows a sequence of operations of a device according to an embodiment of the invention allowing the release of cells of a first type, and Figure 3D-Figure 3E showing the sequence of operations of the device allowing the release of cells of the second type;

[0058] - FIG. 4A to FIG. 4E shows a sequence in which a device according to an embodiment of the present invention allows for simultaneous capture of multiple cell pairs;

[0059] - Figure 5 shows a perspective view of an apparatus according to an embodiment of the present invention;

[0060] - Figure 6 Shown in open position Figure 5 The device traps;

[0061] - Figure 7 Shown in closed position Figure 6 The device trap.

[0062] - Figure 8 Cell capture trap and cell pairing are shown. a) SEM image shows a close-up view of a trap with two capture sites. The figures correspond to the capture sites of the innovative device. b) Brightfield (top left) and fluorescence (top right) views of paired immune cells (white, Fura2-AM calcium probe) and leukemia cells (dark, DiI staining). Pairing occurs regardless of cell size (bottom).

[0063] - Fig. 9 shows how to monitor cell activity in cells. Ca 2+ Imaging experiments show the activity (Ca) of immune cells (natural killer cells, NK92 cells) after forming immune synapses (IS) with leukemia cells (K562 leukemia cells). 2+ mobilization);

[0064] - Fig.10 Schematically illustrates an embodiment of a device including an electrode array configured to generate bubbles to selectively retrieve single cells, trapped cell pairs, or cell triplets from a trap;

[0065] - FIG. 11A to FIG. 11C An embodiment of an electrode array is schematically shown. DETAILED DESCRIPTION

[0066] In this context, the inventors hereby present a specific microfluidic cell pairing device that has been built to address these needs, namely, to study cell-cell interactions between primary cells and their interacting partners (e.g., primary cancer cells and lymphocytes).

[0067] The inventors' device includes a microfluidic channel adapted to a unidirectional flow of a contained solution in order to dynamically adapt its geometry to the size of a patient's target cells, thereby capturing cells of different sizes, such as lymphocyte-cancer cell pairs. This adjustable cell pairing device overcomes the limitations of current cell pairing systems in managing differences in cell sizes and cell types.

[0068] By making the capture structure movable and activating the structure, the inventors can adjust the capture zone size of different cell types in real time to optimize cell pairing. Therefore, no pressure is applied to different cell types. In addition, after pairing, the activated trap can mechanically separate cells and release cells according to type. Finally, it is preferred to pair cells in parallel at multiple sites (on average about 100) to increase the statistical relevance of the detection.

[0069] As described below, the device enables analysis of molecular events occurring at a synapse between two cells as well as molecular events occurring within a cell during the interaction of two cells, for example using live imaging or confocal microscopy. It also allows for stimulation of captured cells with candidate molecules or identification of specific biomarkers that may predict disease outcome or sensitivity to treatment.

[0070] Finally, and importantly, the device of the present invention enables the retrieval of individual cells after synapse formation in order to expand these cells in vitro with the goal of further analyzing the cells or, possibly, transferring them back into the patient after processing them.

[0071] The device of the present invention comprises a microfluidic channel adapted for unidirectional flow of a solution containing cells. The solution may contain a mixture of at least two different types of cells; or, two solutions may flow sequentially in the microfluidic channel, one solution containing a first type of cells and the other solution containing a second type of cells.

[0072] The first type of cells and the second type of cells can be the same or different. In some embodiments, the first type of cells have a different size than the second type of cells; in the examples developed below, the first type of cells have a smaller size than the second type of cells.

[0073] At least two adjustable traps are arranged in the microfluidic channel.

[0074] The first trap is configured to capture cells of a first type and the second trap is configured to capture cells of a second type. Generally, each trap is configured to capture a cell of a corresponding type. However, in some embodiments (see the detailed description below), Figure 2A-2F ), a trap can be configured to capture two cells to form a cell triplet.

[0075] These traps can have different heights (height is a dimension perpendicular to the bottom of the microfluidic channel). For example, the larger the cell being trapped, the taller the trap.

[0076] The traps may not be arranged at the same position in the microfluidic channel. Specifically, in particular if the first trap is intended to capture smaller cells than the second trap, the first trap may be located upstream of the second trap along the flow direction of the solution, the distance along said direction being selected to allow interaction between cells captured in the first trap and the second trap.

[0077] Each trap comprises a pair of fingers arranged in a microfluidic channel, at least one of the fingers being coupled to a respective actuator. The actuator is adapted to move one of the two fingers so as to adjust the first trap in a direction transverse to the flow between an open position allowing the first cell to pass between the fingers and a closed position adapted to the size of the cell to be captured so as to allow the cell to be captured between the fingers. Having only one finger coupled to the actuator allows the design of the device to be simplified. However, having two fingers coupled to respective actuators allows the versatility of the device to be increased, for example by allowing the entire trap to be displaced in a transverse direction. In particular, it may be advantageous to initially offset the first trap and the second trap from each other in a transverse direction to help capture the first cell and the second cell in the respective traps, and then to move the first trap and the second trap relative to each other in a transverse direction so as to align them along the direction of the flow and form a cell pair.

[0078] In this context, "capture" means retaining the cell in the trap, preferably by applying minimal restraint to the cell with the fingers so that the cell remains substantially free. To this end, the trap does not need to be completely closed, i.e., with the fingers in close contact. Even in the closed position, the fingers may be separated by small gaps, as long as the gaps are smaller than the size of the cell so that the cell cannot pass through the gaps.

[0079] The first trap and the second trap are arranged relative to each other so that when the first trap and the second trap are in a closed position, a cell pair including a trapped first cell and a second cell is formed, wherein the first cell and the second cell can interact with each other.

[0080] Figures 1A to 1E The operation of the first trap and the second trap is schematically shown as seen from the top of the microfluidic channel.

[0081] Figure 1A The fingers 10a, 10b of the first trap 1 and the fingers 20a, 20b of the second trap 2 are shown in the open position. The direction of flow is indicated by arrows F.

[0082] Figure 1B The fingers 10a, 10b of the first trap 1 are shown in the closed position, while the fingers 20a, 20b of the second trap 2 are still in the open position. To reach the closed position, the fingers 10a, 10b are moved towards each other in a direction transverse to the flow F by respective actuators (not shown).

[0083] Figure 1C Representation and Figure 1B Same configuration, with the first cell C1 trapped in the first trap 1.

[0084] Figure 1D The first trap 1 is shown still in the closed position, in which the first cell C1 is trapped, and the second trap 2 is also in the closed position. To reach the closed position, the fingers 20a, 20b are moved towards each other in a direction transverse to the flow F by respective actuators (not shown).

[0085] Figure 1E Representation and Figure 1D The same configuration, where a first cell C1 is captured in a first trap 1 and a second cell C2 is captured in a second trap 2. The first and second traps 1, 2 are close enough to each other to form a cell pair of cells C1 and C2, i.e., even if cells C1, C2 are not in physical contact with each other, interaction between cells C1 and C2 is allowed. The size and arrangement of the traps can be designed by a skilled person according to the size of the cells to be captured and the type of interaction to be observed.

[0086] Of course, the present invention is not limited to capturing a pair of cells, but can also be applied to capturing triplets of cells, or even a group of more cells. In this case, the number of traps or the configuration of the traps can be adjusted to allow the desired cell group to be constructed.

[0087] Cell triplets can be captured in a variety of ways.

[0088] In some embodiments, a third trap (not shown) is added, which has the same design as the first and second traps, i.e., two fingers and at least one actuator. The third trap can be aligned with the first and second traps along the flow direction. The size of the third trap is adjusted to the size of the cells to be captured.

[0089] In other embodiments, Figure 2A-2F As shown, one of the first trap and the second trap (in this case, the first trap) is designed to capture two cells. In this way, there is no need to add a third trap different from the first trap and the second trap to capture the third cell, which will increase the complexity of the device.

[0090] Figure 2A The fingers 10a, 10b of the first trap 1 are shown in the closed position, whereas the fingers 20a, 20b of the second trap 2 are in the open position. The flow direction is indicated by arrows F.

[0091] Figure 2B Representation and Figure 2A The same configuration of Trap 1 and Trap 2, where the first cell C1 is trapped in the first Trap 1.

[0092] Figure 2C = indicates that the second trap 2 is still in the open position. By moving the finger 10b away from 10a, the first trap 1 has been partially reopened, while still retaining the first cell C1. The opening width of the first trap 1 is adapted to capture an additional cell C3, such as Figure 2D As shown. Cell C3 may be of the same type as cell C1 or of a different type. Advantageously, cells C1 and C3 have substantially the same size.

[0093] Figure 2E Representation and Figure 2D Identical configuration of the first trap 1, where cells C1 and C3 are trapped in trap 1, and the second trap 2 in the closed position.

[0094] Figure 2F Representation and Figure 2E The first trap and the second trap are configured identically, wherein the cell C2 is captured in the second trap 2. Thus, cells C1, C2 and C3 form a cell triplet.

[0095] This trapping device is particularly advantageous because it not only allows the formation of cell pairs or cell triplets, but also allows the easy release of trapped cells once cell-cell interactions are observed without changing the flow in the microfluidic channel. In contrast, in the case of fixed traps, releasing trapped cells is rather complicated and usually requires the solution to flow in the opposite direction to the flow F in order to force the cells out of the trap.

[0096] Figure 3A It indicates that trap 1 and trap 2 are in a closed position, wherein a pair of first and second cells C1, C2 are captured in the corresponding traps.

[0097] like Figure 3BAs shown, the second trap 2 is moved away from the first trap 1 in a direction transverse to the flow F, the second trap 2 still being in a closed position. Thus, both trap 1 and trap 2 are closed but offset from each other in a transverse direction. This displacement causes the first cell to be separated from the second cell.

[0098] like Figure 3C As shown, the first trap 1 is opened by moving at least one of the fingers 10a, 10b by a corresponding actuator. As a result, the cell C1 is released and leaves the trap 1 in the direction of the flow F through the opening between the fingers 10a, 10b.

[0099] Figure 3D = represents trap 1 and trap 2 in a closed position, wherein the first and second cell pairs C1, C2 are trapped in the respective traps. The second trap 2 is moved away from the first trap 1 in a direction transverse to the flow F, the second trap 2 still being in a closed position. Thus, both trap 1 and trap 2 are closed, but offset from each other in a transverse direction. This displacement causes the first cell to be separated from the second cell.

[0100] like Figure 3E As shown, the second trap 2 is opened by moving at least one of the fingers 20a, 20b by a corresponding actuator. As a result, the cell C2 is released and leaves the trap 2 in the direction of the flow F through the opening between the fingers 20a, 20b without disturbing the first trap 1.

[0101] certainly, Figure 3A-3C and Figure 3D-Figure 3E The sequence shown can also be carried out simultaneously, namely by moving the closed traps away from each other in the direction transverse to the flow and opening the first trap and the second trap simultaneously.

[0102] like Figure 3E As shown, at least one of the fingers 20a, 20b is moved by a corresponding actuator to open the second trap 2. As a result, the cell C2 is released and leaves the trap 2 through the opening between the fingers 20a, 20b in the direction of the flow F. Since the first trap 1 is offset from the second trap 2 in the lateral direction, the first trap does not hinder the cell C2 from flowing with the solution.

[0103] Thus, the device can be prepared again to capture a new pair of cells.

[0104] although Figures 1A to 3E The operation of the device is demonstrated for capturing one cell pair or cell triplets, but the device can certainly be designed to capture multiple cell pairs or cell triplets simultaneously.

[0105] For example, Figure 4AAs shown, the device may include a plurality of first traps 1 (e.g., five first traps) and a plurality of second traps 2 (e.g., five second traps). As previously described, each first trap 1 includes a finger 10a and a finger 10B, which are movable relative to each other in a direction transverse to the flow F. Preferably, in order to simplify the design and operation of the device, all fingers 10a are coupled to the same first actuator (not shown), and all fingers 10b are coupled to the same second actuator (not shown). Similarly, each second trap 2 includes a finger 20a and a finger 20B, which are movable relative to each other in a direction transverse to the flow F. Preferably, in order to simplify the design and operation of the device, all fingers 20a are coupled to the same first actuator (not shown), and all fingers 20b are coupled to the same second actuator (not shown).

[0106] exist Figure 4A Preferably, each first trap is aligned with the corresponding second trap in the direction of flow F.

[0107] like Figure 4B As shown, the first traps 1 are closed simultaneously. Preferably, the two fingers of each first trap 1 are moved towards each other by the same distance to keep the first trap aligned with the second trap.

[0108] like Figure 4C As shown, cell C1 is captured in each first trap 1 .

[0109] like Figure 4D As shown, the second trap 2 is closed at the same time. Preferably, both fingers of the second trap 2 move towards each other by the same distance to keep the second trap aligned with the first trap.

[0110] like Figure 4E As shown, the cell C2 is captured in each first trap 2 , thereby forming a cell pair with the cell C1 captured in the corresponding first trap 1 .

[0111] Once cell pairs are observed, they can be Figure 3A-3E The device is operated to release the first cell simultaneously and the second cell simultaneously. Alternatively, all cell pairs can be released simultaneously by opening all traps.

[0112] The actuator is controlled by a control unit.

[0113] The control unit is configured to receive sizes of the first cell and the second cell, and control the first actuator and the second actuator to adjust the gaps between the fingers of the first trap and the gaps between the fingers of the second trap in the closed position of the first trap and the second trap, respectively, based on the received sizes of the first cell and the second cell, the gaps being smaller than the corresponding cells, thereby retaining the cells in the traps.

[0114] In some embodiments, the sizes of the first cell and the second cell can be manually input by a user, such as via a user interface coupled to the control unit.

[0115] In other embodiments, the device may include a measuring unit coupled to the control unit, the measuring unit being adapted to measure in real time the size of the first cell and the second cell in the solution flow. Thus, the control unit receives the measured size of the first cell and the second cell in real time and may adjust the operation of the actuator accordingly to accommodate the gap between the fingers in the closed position.

[0116] In some embodiments, the device can include at least one mechanical sensor or electrical sensor adapted to detect entrapment of a cell in a trap.

[0117] For example, in particular, a mechanical sensor can detect the mechanical force exerted by a cell on at least one finger of the trap. Mechanical sensing can be achieved by measuring the resonant frequency of the actuator and determining the number of trapped cells by the change in the frequency of the actuator. Mechanical sensing can also be achieved by static measurements, where one actuator can be displaced to compress the cell. The displacement induced by the cell rigidity on the second actuator is due to the presence of the cell. The number of cells can therefore be assessed by the displacement of the second actuator.

[0118] The electrical sensor can detect the current flowing between the trapping fingers passing through the cells. Electrical sensing can be achieved by measuring the conductivity between the actuator pairs with and without trapped cells and determining the number of trapped cells based on the decrease in conductivity, which is due to the fact that the conductivity of cells is lower than that of the solution.

[0119] The device can be used in a system for analyzing real-time interactions of at least one cell pair or cell triplets.

[0120] The system includes optical sensors, electrical sensors, mechanical sensors and / or chemical sensors configured to acquire data related to the interaction of a pair of cells or a trio of cells captured by the device. Electrical sensors and mechanical sensors can be used not only to detect the presence of the above cells, but also to determine the number of cells captured and measure the mechanical and / or electrical properties of the captured cells. Optical sensors and chemical sensors can detect fluorescent imaging of cell pairs and detect and quantify cell interactions, such as the activation of an immune response between these cells.

[0121] Figure 5 A perspective view of a device according to an embodiment of the present invention is shown.

[0122] The device comprises a microfluidic channel 3 , in which the flow direction of the solution containing cells is indicated by arrow F.

[0123] like Figure 6 and Figure 7 As better shown, the device comprises two rows of traps, each row comprising three first traps 1 and three second traps 2 . Figure 6 The trap is shown in the open position, while Figure 7 The trap is shown in the closed position.

[0124] As described above, each first trap 1 includes the finger 10 a and the finger 10 b , and each second trap 2 includes the finger 20 a and the finger 20 b .

[0125] The device comprises four actuators 11a, 11b, 21a, 21b arranged on both sides of the trap. Each actuator may be a programmable stepper motor. For example, the actuator may be a piezoelectric linear motor.

[0126] Actuator 11a is configured to move finger 10a of each first trap, actuator 11b is configured to move finger 10b of each first trap 1, actuator 21a is configured to move finger 20a of each second trap, and actuator 21b is configured to move finger 20b of each second trap.

[0127] To this end, each of the fingers 10a, 10b, 20a and 20b is coupled to a respective actuator 20a, 20b, 21a, 21b via a respective rod 12a, 12b, 22a, 22b. The rods 12a, 12b, 22a, 22b extend substantially perpendicular to the flow F.

[0128] Therefore, for each row of traps, the fingers 10a of the three first traps 1 are supported by the rod 12a and can be moved simultaneously by the actuator 11a, and the fingers 10b of the three first traps 1 are supported by the rod 12b and can be moved simultaneously by the actuator 11b; similarly, the fingers 20a of the three second traps 2 are supported by the rod 22a and can be moved simultaneously by the actuator 21a, and the fingers 20b of the three second traps 2 are supported by the rod 22a and can be moved simultaneously by the actuator 21b.

[0129] Advantageously, the device comprises two pairs of beams 13a, 13b, 23a, 23b extending along the flow F and connected to one of the rods 12a, 12b, 22a, 22b and to the respective actuator 11a, 11b, 21a, 21b. The rods extend in a direction substantially perpendicular to the beams.

[0130] Each end of the beam is fixed relative to the microfluidic channel, but each beam can be deformed in a direction transverse to the flow F by a corresponding actuator 11a, 11b, 21a or 21b, thereby moving the corresponding rod 12a, 12b, 22a or 22b in a direction transverse to the flow F to adjust the first trap or the second trap.

[0131] To minimize assembly, each beam can be integrally formed with the corresponding rod and finger. For example, the beam, rod and finger can be made of photopolymer resin. To this end, methods known in the field of microelectronics can be used.

[0132] Of course, the invention is not limited to the embodiment shown and can be implemented with different embodiments of the actuator and the mechanical transmission between the actuator and the finger. Furthermore, the number of traps can be adjusted according to the needs of the experiment.

[0133] In some embodiments, a selected single captured cell or captured cell assembly (cell pair or cell triplets) can be released by generating bubbles in the solution surrounding the trap by electrolysis of the solution. To this end, the device includes a plurality of electrically isolated electrodes at the bottom of the microfluidic channel, which are arranged in an array. The nodes of the array (i.e., the area where two electrodes overlap) are located below the trap. In this way, when a sufficient potential difference is applied between the two overlapping electrodes (i.e., the potential difference is greater than the potential difference that triggers electrolysis of the solution), bubbles are generated at the bottom of the microfluidic channel and rise in a substantially vertical direction in the solution, thereby pushing the cell or cell assembly out of the trap. The released cell or cell assembly can then be driven along the microfluidic channel by the flow of the solution.

[0134] Fig.10 The electrode array arranged on the bottom 30 of the microfluidic channel is schematically shown. The first set of electrodes E11, E12, E13 extends perpendicular to the direction of the flow F, while the second set of electrodes E21, E22, E23 extends parallel to the direction of the flow F and thus perpendicular to the first set of electrodes E11, E12, E13. Each electrode in the first set crosses an electrode in the second set below the corresponding trap. "Below" here means that the intersection or overlapping area is aligned with the capture zone along a line perpendicular to the bottom of the microfluidic channel.

[0135] The first set of electrodes and the second set of electrodes are electrically isolated from each other.

[0136] Each electrode is connected to a power supply configured to selectively apply a determined potential to each electrode. Thus, a potential difference may be generated at each intersection region.

[0137] Depending on the potential applied to each electrode, the potential difference may be greater than the potential threshold that allows electrolysis of the solution flowing in the microfluidic channel. In this case, bubbles are generated in the intersection area and rise in the solution, thereby pushing the cell or cell assembly out of the corresponding trap. Thus, the cell or cell assembly can be retrieved from the trap without activating any actuator to move the fingers away from each other.

[0138] In contrast, if the potential difference is less than the potential threshold, no electrolysis occurs and the cell assembly remains in the trap.

[0139] For example, in Fig.10 In the embodiment shown, a 3V potential is applied to electrodes E11, E13, E21, and E23, a 1V potential is applied to electrode E12, and a 5V potential is applied to electrode E22. As a result, a potential difference ΔP1=0V is applied to the intersection regions of electrodes E13 and E21 and between electrodes E13 and E23; a potential difference ΔP2=2V is applied to the intersection regions of electrodes E12 and E21 and between electrodes E11 and E22; and a potential difference ΔP3=4V is applied to the intersection region between electrodes E12 and E22. Of course, the number of electrodes in each group and their relative arrangement are for illustration purposes only and are not intended to be limiting.

[0140] Assuming that a potential difference of at least 3 V is required to produce electrolysis of the solution, the potential differences ΔP1 and ΔP2 are too low to produce electrolysis in the corresponding intersection regions; however, since ΔP3 is greater than 3 V, electrolysis occurs in the intersection region between electrodes E12 and E22. As a result, a bubble B is formed in the intersection region, and the bubble B rises in a substantially vertical direction, pushing the cell pair C1 and C2 out of the trap.

[0141] The applied potential difference depends on the electrode material, electrode geometry and solution (buffer or medium) ionic strength and can be up to 20 V DC. An AC signal (up to 1 or 2 MHz) can also be applied for dielectrophoresis or AC electroosmosis to release cells, cell pairs or cell triplets.

[0142] The electrode array can be made by patterning an electrically conductive material (e.g., indium tin oxide (ITO), or a metal such as gold) to form a first set of electrodes (e.g., a set of parallel electrodes) at the bottom of the microfluidic channel. The electrode thickness can be up to 500 nm. These electrodes are covered with a first dielectric layer, the thickness of which is greater than the thickness of the electrodes to electrically isolate the electrodes. The dielectric material can be, for example, SiO2, spin-on glass, or CYTOP TM (A fluorinated polymer).

[0143] The second set of electrodes is patterned perpendicular to the first set of electrodes and then covered with a second dielectric layer having a thickness greater than that of the electrodes so as to electrically isolate each electrode. The width of each electrode is of the same order of magnitude as the size of the cell to be captured, for example a few micrometers.

[0144] The dielectric layer is then etched around the overlapping electrode regions to form recesses wherein at least a portion of each electrode is exposed to a solution flowing in the microfluidic device. Figures 11A-11C Various embodiments of such grooves are schematically shown.

[0145] Fig.11AA portion of the bottom of a microfluidic device according to an embodiment is shown, including a portion of a first electrode E11 and a portion of a second electrode E21 that overlaps the first electrode portion. As described above, the electrodes E11 and E21 are embedded in the first dielectric layer and the second dielectric layer (here represented as a single dielectric layer 31). A groove 300 is formed through the dielectric layer 31 to the bottom 30 of the microfluidic channel. Fig. 11B (Right now Fig.11A The partial cross-sectional view of the groove clearly shows that the etching partially exposes the upper surface and side surfaces of the second electrode E21 and the upper surface and side surfaces of the first electrode E11 on both sides of the overlapping second electrode E21. However, the etching does not remove the dielectric material between the first electrode and the second electrode.

[0146] The groove may have a circular shape, e.g. Figure 11A-11B As shown, but it can have any suitable shape, such as a rectangle or square (such as Fig. 11C In the embodiment shown, the groove is located in the center of the overlapping area, but it can also be placed in different positions as long as at least a portion of the upper surface and / or side of each electrode is exposed.

[0147] Fig. 11C FIG. 4 shows a portion of the bottom of a microfluidic device according to another embodiment, including a portion of a first electrode E11 and a portion of a second electrode E21 that overlaps the first electrode portion. Fig.11A As shown, electrodes E11 and E21 are embedded in the first dielectric layer and the second dielectric layer (here shown as a single dielectric layer 31). A groove 300 is formed through the dielectric layer 31 to the bottom 30 of the microfluidic channel. Fig.11A In contrast to the embodiment of the present invention, etching only exposes the side surfaces of the first and second electrodes E11, E21. In this embodiment, the groove is thus formed along one side surface of the electrode E11 and the electrode E21. As described above, etching does not remove the dielectric material located between the first and second electrodes.

[0148] The grooves allow the solution to contact the electrodes only at or near the overlapping area. Each groove is formed directly below the trap, thereby allowing the formation of a cell assembly.

[0149] The size of the grooves (e.g., the diameter of a circular groove, or the length / width of a square or rectangular groove) is selected to produce bubbles of a size suitable for propelling cells or cell assemblies. To this end, the size of the grooves (and the size of the bubbles produced thereby) is equal to or slightly larger than the size of the cells, which can be of the order of 10 μm.

[0150] In addition, the device of the present invention can be used not only to study the interaction between the above-mentioned immune cells, but also to study any cell-cell interaction, i.e., physical or chemical interaction between any kind of cells, wherein the any kind of cells are: cells of different sizes, cells of different sources, cell line cells or primary cells, naturally occurring cells or cells produced by recombinant generation. Specifically, these cells can be circulating cells, such as blood cells (e.g., red blood cells, platelets, granulocytes, agranulocytes, etc.), urine cells, sperm cells, adipocytes (white or brown adipocytes), metastatic or circulating tumor cells, etc., or tissue-related cells, such as epithelial cells, fibroblasts, egg cells, solid cancer cells, skin cells (keratinocytes, melanocytes, Merkel cells or Langerhans cells), muscle cells (skeletal muscle, cardiac muscle or smooth muscle cells), nerve cells (e.g., neurons or glial cells), endothelial cells, pancreatic cells, chondrocytes, bone cells (e.g., osteoblasts, osteoclasts, osteocytes), etc. They can be differentiated cells or stem cells. Cells can be prokaryotic cells (e.g., bacteria) or eukaryotic cells (including yeast cells).

[0151] To be properly captured in the device of the invention, the cells to be studied preferably have an average diameter greater than about 0.5 μm. However, in specific embodiments, the device of the invention can be used to analyze the behavior of these cells when in contact with smaller microorganisms (e.g., bacteria, viruses, protozoa, etc.).

[0152] The device of the present invention can tolerate a certain degree of cell aggregation. However, in specific embodiments, the cell concentration in the sample to be analyzed should be adjusted so that there is no or little cell aggregation. For example, the cell concentration in the solution flowing in the device of the present invention is preferably less than 500,000 cells per milliliter. Higher concentrations can be diluted in the culture medium required for the survival of such cells.

[0153] Cells are preferably isolated from a blood sample prior to insertion into the device. For example, blasts used in the experiments described below were isolated from peripheral blood after separation by Ficoll and removal of lymphocytes using immunomagnetic negative selection (CD3 for T lymphocytes and CD20 for B lymphocytes).

[0154] Specifically, solutions that will flow in the channel (such as culture medium and other solutions, such as plasma) can be filtered so that they do not contain cells or other components with a diameter greater than 2 μm (when the diameter of the cells being studied is greater than 5 μm) or greater than one-third of the diameter of the cells being studied (when the diameter of the cells is less than 5 μm).

[0155] Once the target cell is captured in the device of the present invention, a variety of imaging techniques can be used to analyze the behavior of the cell. Imaging is the most powerful tool for visualizing the structure of cells and their behavior in vivo. So far, many microscopy-based imaging methods have been developed, such as confocal microscopy, super-resolution microscopy, two-photon microscopy, electron microscopy, and atomic force microscopy. Imaging can use labeled antibodies to quantify proteins, RNA, lipids, and sugar chains.

[0156] Analysis of real-time interactions of captured cell pairs or cell triplets can include exposing the cells to solutions with specific properties (e.g., pH or viscosity) to mimic cell interactions under specific conditions. Thus, one or more solutions with different properties can flow in a microfluidic channel to selectively contact specific cells or collectively contact the entire captured cell pair or cell triplet.

[0157] In some embodiments, selected cells or entire captured cell pairs or cell triplets can be exposed to solutions with different pH values, particularly acidic pH values ​​(e.g., about 6.2 to 6.5) that are lower than the pH found in normal tissues (about 7.4). This can explain the changes found in vivo or in non-healthy conditions (acidic microenvironment in cancer tissue or bone marrow).

[0158] In other embodiments, selected cells or entire captured cell pairs or cell triplets can be exposed to solutions having various viscosities, such as solutions that simulate cell interactions in blood (thus, the viscosity of the solution is 3 to 5 cP (3-5 mPa.s), corresponding to average blood viscosity) and / or bone marrow (thus, the viscosity of the solution is 30 to 38 mPa.s, corresponding to average bone marrow viscosity).

[0159] Once the target cells are released from the device of the present invention, other molecular tools can be used to decipher the molecular signaling pathways that are altered by cell-cell interactions. For example, flow cytometry can quantitatively and high-throughput analyze single cells. Multiple labeling identifies the composition of various cell types based on the expression of surface protein markers. In addition, flow cytometry can be used to quantitatively analyze multiple proteins in single cells using dyes or fluorescently labeled antibodies. Similarly, flow cytometry has also been used to measure the expression levels of mRNA or microRNA using labeled antisense nucleotides. Combined with fluorescence-activated cell sorting (FACS), target cells can be separated and collected in a high-throughput manner, enabling further comprehensive gene expression and proteomic analysis. Imaging flow cytometry is another high-throughput method that analyzes thousands of single cells per second by simultaneously taking cell images to provide morphological information. Recent advances in droplet-based technology have made transcriptome analysis of single cells possible. Combined with DNA barcoding, thousands of single cells can now be analyzed. Since the amount of transcribed protein and actual protein is usually different, proteome analysis needs to be performed in single cells. Although it is not yet possible to perform a comprehensive analysis of proteins, mass cytometry is one of the highest throughput protein analyses. Isolated cells are labeled with multiple metal-conjugated antibodies, and the amount of target protein is then detected by time-of-flight flow cytometry (CyTOF) mass spectrometry. Current technology is capable of measuring approximately 40 proteins simultaneously. Imaging-based mass spectrometry (IMS) is a method that integrates the strengths of imaging and unbiased proteome analysis through matrix-assisted laser desorption ionization. By measuring the mass / charge signal at each coordinate, it can analyze the presence of target molecules and their localization information in tissues. IMS is able to analyze thousands of proteins, lipids, and metabolites directly from tissues without labeling. Other molecular tools are described in Nishida-Aoki N. and Gujral TS, Emerging approaches to study cell-cell interactions in tumor microenvironment, Oncotarget, 2019, 10(7): 785-797.

[0160] In a preferred embodiment, the device of the present invention is used to study the immune synapse (IS), i.e., the cell pair involving an immune cell and its target cell. Functional analysis at the level of the immune synapse (IS) is crucial to improve the efficiency of immunotherapies currently used in the clinic. In fact, although immunotherapies targeting advanced cancers have shown very promising results, most patients do not respond to a specific treatment. In this context, it is very important to identify relevant biomarkers that can predict the efficiency of these therapeutic molecules.

[0161] The immune synapse involves an antigen-presenting cell (e.g., a B cell, dendritic cell, macrophage, virus-infected cell, or cancer cell) and a target cell, such as a CD4 + T lymphocytes, CD8 + T lymphocytes, B lymphocytes or natural killer cells (NK cells). Specifically, the device of the present invention can be used to study the specific interaction between dendritic cells and T cells, or between tumor cells and T cells.

[0162] In a specific but preferred embodiment, the device of the invention is used to dissect the contribution of cell-intrinsic (i.e. T cell parameters) and cell-extrinsic (i.e. cancer cell parameters) factors during the immune synapse reaction in the clinical context of cancer. The device of the invention actually represents a very valuable tool in the development of precision medicine, allowing the study of the immune synapse between T cells and cancer cells, the restoration of T lymphocyte activity, or the understanding of the molecular mechanisms of resistance to therapeutic molecules.

[0163] Although T lymphocytes are generally associated with the elimination of tumor cells, inhibitory signaling pathways that target immune effector cells and tumor cells can lead to tumor dormancy and / or tumor escape (B. Quesnel, Acta Pathologica, Microbiologica, et Immunologica Scandinavica, Vol. 116, No. 7-8, pp. 685-94, 2008). In addition, it is proposed that tumor cells exhibit characteristics that may lead to resistance to T lymphocytes (quiescence, dormancy, stemness...) (B. Quesnel, Acta Pathologica, Microbiologica, et Immunologica Scandinavica, Vol. 116, No. 7-8, pp. 685-94, 2008; Y. Touil et al., Scientific reports, Vol. 6, p. 30405, 2016). Therefore, understanding the dialogue between T cells and tumor cells is crucial to preventing relapse in patients.

[0164] Thanks to the device of the present invention, as described above, it is possible to detect the presence of Ca by various conventional methods (e.g., transcriptomics, proteomics, genomics) and more precisely by examining the Ca 2+ Signal transduction (see Figure 8 ), checkpoint inhibitory proteins, or expression of stemness markers (e.g., by confocal microscopy, Western blotting, and / or flow cytometry) to visualize the dialogue between T cells and tumor cells.

[0165] The preferred analytical tool is calcium signaling. It has been well established that calcium signaling regulates T cell activity (M. Trebak and J. P. Kinet, Nature reviews Immunology, Vol. 19, No. 3, pp. 154-169, 2019). In addition, calcium signaling is associated with all markers of cancer (N. Prevarskaya, R. Skryma and Y. Shuba, Physiological reviews, Vol. 98, No. 2, pp. 559-621, 2018).

[0166] The device of the present invention is well suited for functional studies of immune synapses formed by primary cells of different sizes from patients, despite limited access to rare but critical dysfunctional T cells or dormant tumor cells from patients.

[0167] Thus, in preferred embodiments, the device of the present invention is able to decipher calcium signatures at the level of the immune synapse in the context of tumor dormancy.

[0168] In a specific aspect, the present invention provides:

[0169] - A system that can simultaneously perform functional analysis of two cell types (T cells and tumor cells from the same patient) during the formation of the immune synapse and follow the subsequent cellular events.

[0170] - A system that allows to contact rare cells of low frequency and to carry out functional studies, thus enabling to analyze the formation of immune synapses even though it is a rare event in vivo. In fact, only very small amounts of cells are required to carry out experiments using the device of the invention.

[0171] - A system that allows screening of therapeutic molecules or candidate molecules capable of i) reactivating T lymphocytes and evaluating their efficacy against tumor cells at different stages of the disease, or ii) sensitizing drug-resistant cancer cells to T cells.

[0172] These studies can be performed in a clinical setting to assess the activity of T lymphocytes against tumor cells (calcium response, lysis) at major stages of disease (diagnosis, dormant or minimal residual disease, relapse).

[0173] Can be through Ca 2+ Imaging and confocal microscopy to assess Ca2+ associated with IS formation 2+ Signaling and the resulting cellular events. Ca signaling can be studied using specific inhibitors, agonists, antagonists, anti-inhibitory checkpoints (antibodies), and therapeutic molecules or candidate molecules. 2+ Uncovering these regulatory mechanisms provides new perspectives for immunotherapy or helps optimize current immunotherapy regimens.

[0174] Once the cells are released from the trap, further analysis can be performed on the released cells. Cells of the same class (e.g., only T cells or only tumor cells) can then be advantageously collected specifically by sorting them using flow cytometry. Conventional molecular analysis (immunostaining, western blot, PCR, spectroscopy, proteomics, etc.) can then be performed to identify specific genomic or proteomic features or biomarkers in these cells.

[0175] Having studied the immune synapse, the released T cells and resistant tumor cells are analyzed, potentially providing a specific genomic signature for each stage of cancer disease or for each patient. In this case, the device of the invention enables the discovery of new biomarkers predicting disease outcome.

[0176] Example

[0177] The specific protocol for using the device of the present invention is as follows: Prior to cell loading, isolated NK cells and leukemia K562 cells were cultured for 24 hours to remove the staining antibodies used for sorting according to their phenotype. Cancer cells have been stained with a viability / tracing dye (DiI). Single cell Ca in the IS has been monitored using SEM. 2+ Signal transduction (see Figure 8 and Fig. 9 ). Ca 2+ The sensitive Fura-2 dye allows real-time monitoring of cytoplasmic Ca 2+ concentration. 2+ The signature (without stimulation) was compared to that observed with NK cells.

[0178] This protocol allows the assessment of intracellular Ca during IS formation. 2+ level changes, but it can also be used to monitor:

[0179] - cytotoxic lysis,

[0180] - resistance to lysis,

[0181] - Effects of IFN-g and TNF-α (cytokine) release on calcium responses in IS,

[0182] -etc.

[0183] Alternatively, T cells can be used instead of NK cells and activated with HLA-DR+ cancer cells pre-incubated with different concentrations of superantigens or IS pre-coated with different concentrations of anti-CD3 antibodies. The ratiometric Fura-2 dye can then be loaded into the cells for Ca 2+ Signal transduction studies.

[0184] Specifically, the involvement of Ca in IS can be assessed 2+ Expression and localization of key proteins involved in IS-induced Ca signaling, cytotoxic activity, apoptosis, proliferation, and cytokine (IFN-g, TNF-α) release. 2+ These inhibitors will be added directly to the microfluidic system based on the molecular nature of the channel that responds. Modulation of calcium signaling in the IS using selective agonists, antagonists and therapeutic molecules or candidate molecules will enable the assessment of potential restoration of T lymphocyte function (proliferation, cytokine secretion, cytotoxic lysis) as well as the proliferation / quiescence status of cancer cells.

Claims

1. A device for capturing at least one cell pair in a solution, the solution containing at least one first cell of a first type (C1) and at least one second cell of a second type (C2), the device comprising: - a microfluidic channel (3) adapted for unidirectional flow of a solution (F); - a first trap (1) comprising a pair of first fingers (10a, 10b) arranged in a microfluidic channel (3), at least one of the first fingers (10a, 10b) being coupled to a respective first actuator (11a, 11b), the first actuator being configured to adjust the first trap (1) in a direction transverse to the flow (F) between an open position allowing the passage of a first cell between the first fingers (10a, 10b) and a closed position adapted to the size of the first cell to allow the capture of the first cell between the first fingers (10a, 10b); a second trap (2) comprising a pair of second fingers (20a, 20b) arranged in the microfluidic channel (3), at least one of the second fingers (20a, 20b) being coupled to a respective second actuator (21a, 21b), the second actuator being configured to adjust the second trap (2) in a direction transverse to the flow (F) between an open position allowing the passage of the second cell between the second fingers (20a, 20b) and a closed position adapted to the size of the second cell to allow the capture of the second cell between the second fingers (20a, 20b); Wherein, the first trap (1) is arranged relative to the second trap (2) so that when the first trap and the second trap are in a closed position, a cell pair comprising a captured first cell and a second cell is formed, so that the second cell physically or chemically interacts with the first cell.

2. Device according to claim 1, wherein in the closed position, the second trap (2) is larger than the first trap (1) so as to allow capturing a second cell larger than the first cell in the second trap.

3. The device according to any one of claims 1 to 2, wherein each first finger (10a, 10b) is coupled to a respective first actuator (11a, 11b), and each second finger (20a, 20b) is coupled to a respective second actuator (21a, 21b).

4. The device according to claim 3, wherein the first actuator and the second actuator (11a, 11b, 21a, 21b) are configured to move the first trap relative to the second trap to separate the first cell from the second cell, and to open the first trap and the second trap to release the first captured cell and the second captured cell in the flow (F).

5. A device according to any one of claims 1 to 4, wherein each first or second finger (10a, 10b, 20a, 20b) is coupled to a respective first or second actuator (11a, 11b, 21a, 21b) via a respective first or second rod (12a, 12b, 22a, 22b) substantially perpendicular to the flow (F).

6. A device according to claim 5, wherein each of the first rod and the second rod (12a, 12b, 22a, 22b) can respectively support at least two first fingers or second fingers (10a, 10a, 10b, 10b', 20a, 20a', 20b, 20b') to form at least two first traps and second traps (1, 1', 2, 2') which can be adjusted simultaneously by the first actuator and the second actuator (11a, 11b, 21a, 21b), respectively.

7. The device according to claim 5 or 6 further comprises two pairs of foldable beams (13a, 13b, 23a, 23b) extending along the flow (F) and connected to one of the first rod and the second rod (12a, 12b, 22a, 22b), each end of the foldable beam being fixed relative to the microfluidic channel (3), wherein each foldable beam can be deformed by a corresponding first actuator or a second actuator (11a, 11b, 21a, 21b) so as to move the corresponding first rod or the second rod in a direction transverse to the flow (F), thereby adjusting the first trap or the second trap.

8. The device according to any one of claims 1 to 7 further comprises a control unit, which is configured to receive the sizes of the first cell and the second cell, and to control the first actuator and the second actuator (20a, 20b, 21a, 21b) respectively based on the received sizes of the first cell and the second cell to adjust the distance between the first fingers and the distance between the second fingers in the closed position of the first trap and the second trap. 9 . The device according to claim 8 , further comprising a measuring unit adapted to measure the sizes of the first cell and the second cell in real time, and the control unit is configured to receive the measured sizes of the first cell and the second cell in real time.

10. The device according to any one of claims 1 to 9, further comprising at least one mechanical sensor or electrical sensor adapted to detect cells that have been trapped in the respective first trap, the second trap and the third trap (if applicable).

11. A device according to any one of claims 1 to 10, wherein at least one of the first actuators is configured to adjust the size of the first trap in the closed position after capturing a first cell to allow a third cell to be captured in the first trap to form a cell triplet with the second cell captured in the second trap, so that the first cell, the second cell and the third cell interact physically or chemically.

12. The device according to any one of claims 1 to 10, further comprising a third trap, the third trap comprising a pair of third fingers arranged in the microfluidic channel (3), at least one of the third fingers being coupled to a corresponding third actuator, the third actuator being configured to adjust the third trap in a direction transverse to the flow (F) between an open position and a closed position, the open position allowing a third cell to pass between the third fingers, the closed position being adapted to the size of the third cell to allow the capture of the third cell between the third fingers; in, The third trap is arranged relative to the first trap and the second trap so as to form a cell triplet containing the captured first cell, second cell and third cell when the first trap, second trap and third trap are in a closed position, so that the first cell, second cell and third cell undergo physical or chemical interaction.

13. A device according to any one of claims 1 to 12, further comprising an array of electrically isolated electrodes (E11, E12, E13, E21, E22, E23), the array being arranged at the bottom (30) of the microfluidic channel so that an overlapping region of two electrodes is located below each trap, at least one surface of each electrode being exposed in a groove (300) in the overlapping region, each electrode being connected to a power supply so as to selectively apply a potential difference to the solution in each overlapping region.

14. A method for capturing at least one cell pair in a solution containing at least one first cell of a first type and at least one second cell of a second type, the method comprising: - causing the solution to flow in a microfluidic channel of a device according to any one of claims 1 to 13; - activating at least one first actuator to close said first trap, the dimensions of said first trap in the closed position being adapted to the dimensions of said first cell; - capturing a first cell in said first trap; - activating at least one second actuator to close said second trap, the dimensions of said second trap in the closed position being adapted to the dimensions of said second cell; - capturing a second cell in the second trap, the second cell forming a cell pair with the first cell, the second cell interacting physically or chemically with the first cell.

15. A method for capturing cell triplets in a solution containing at least one first cell of a first type, at least one second cell of a second type different from the first type, and at least one third cell of a third type, the method comprising: - causing the solution to flow in a microfluidic channel of a device according to any one of claims 1 to 13; - activating at least one first actuator to close said first trap, the dimensions of said first trap in the closed position being adapted to the dimensions of said first cell; - capturing a first cell in said first trap; - activating at least one first actuator to adjust the size of the first trap in the closed position to adapt to the size of the first cell and the third cell; - capturing a third cell in said first trap; - activating at least one second actuator to close said second trap, the dimensions of said second trap in the closed position being adapted to the dimensions of said second cell; - capturing a second cell in the second trap, the second cell forming a triplet with the first cell and the third cell, the first cell, the second cell and the third cell interacting physically or chemically.

16. The method according to any one of claims 14 to 15, implemented using the device according to claim 5, wherein the first trap and the second trap are in an initial open position, wherein the first trap is away from the second trap in a direction transverse to the flow (F), and wherein after the first cell and the second cell are captured in the first trap and the second trap, the first actuator and / or the second actuator are activated to bring the first trap and the second trap closer to each other to produce cell pairs or cell triplets.

17. The method of claim 16, further comprising releasing at least one of the first cell, the second cell, and the third cell (if applicable), by activating the first actuator and / or the second actuator to move at least one of the first trap and the second trap so as to leave the other trap in a direction transverse to the flow (F), and opening at least one of the first trap and the second trap to release the cells captured in the corresponding trap.

18. The method according to any one of claims 14 to 15, implemented using the device according to claim 13, further comprising releasing at least one of the first cell, the second cell and the third cell (if applicable) from a selected trap by applying a potential difference greater than a potential difference that triggers electrolysis, dielectrophoresis or electroosmosis of a solution in a corresponding recess (300) to overlapping electrodes (E11, E21) below the trap so as to generate bubbles (B) adapted to push the first cell, the second cell and / or the third cell out of the trap.

19. A method for analyzing real-time interactions of at least one pair of cells or a trio of cells, comprising: - capturing at least one cell pair in a cell triplet using the method of any one of claims 14 to 16; - using at least one of an optical sensor, an electrical sensor, a mechanical sensor and a chemical sensor, acquiring data related to the interaction of the cell pairs or cell triplets.

20. The method of claim 19, further comprising exposing at least one captured cell pair or cell triplet to a solution having a defined pH value and / or a defined viscosity, the pH value or viscosity being selected to simulate cell interactions under defined circumstances.

21. A system for analyzing real-time interactions of at least one pair of cells or a trio of cells, comprising: - a device according to any one of claims 1 to 13, and - At least one of an optical sensor, an electrical sensor, a mechanical sensor, and a chemical sensor configured to acquire data related to the interaction of a pair or a triplet of cells captured by the device.

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