Apparatus and method for trapping cell pairs and method for analyzing cell pairs in real time - Patents.com

Abstract

Apparatus and method for trapping cell pairs and method for analyzing cell pairs in real time - Patents.com The present invention relates to a device for trapping at least one cell pair in a solution comprising 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 the unidirectional flow of solutions (F); - a first trap (1) arranged in said microfluidic channel (3) and comprising a pair of first fingers (10a, 10b), at least one of said first fingers (10a, 10b) being coupled to a respective first actuator (11a, 11b), said first actuator being configured to adjust said first trap (1) along a direction transverse to said flow (F) between an open position allowing passage of said first cell between said first fingers (10a, 10b) and a closed position adapted to the size of said first cell to allow trapping of said first cell between said 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) along a direction transverse to the flow (F) between an open position allowing passage of the second cell between the second fingers (20a, 20b) and a closed position adapted to a size of the second cell to allow trapping of the second cell between the second fingers (20a, 20b); Here, the first trap (1) is positioned relative to the second trap (2) to form a cell pair comprising the trapped first and second cells, such that the second cell has a physical or chemical interaction with the first cell when the first trap and the second trap are in a closed position.

Description

[Background technology]

[0001] Cell-cell interactions play a crucial role in various biological systems, particularly in immunity, where cell pairings initiate and mediate many important developmental (selection, proliferation, differentiation) and functional (cytolysis, cytokine and antibody production) immune responses (RD Schreiber, LJ Old, and MJ Smyth, Science, vol. 331, no. 6024, pp.). Against this background, it is fundamental to better understand the interaction dynamics between immune cells and their cellular partners.

[0002] These interactions are typically studied by activating cells in bulk cocultures, mixing cell populations, initiating contact with a brief centrifugal co-sedimentation, and stitching together measurements from independent assays performed at different time points. Although such bulk cocultures have revealed important information about these interactions, the results are unfortunately averaged across many different cell types and interaction combinations (B. Dura and J. Voldman, Current opinion in immunology, vol. 35, pp. 23-9, 2015). Such technical approaches mask the intrinsic cellular heterogeneity with respect to interaction potential, variability in antigen-presenting cells, contact time, and all parameters 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 growing evidence that population-wide measurements do not reflect tumor fate by masking the behavior of single cells.

[0003] Moreover, although immunotherapy with immune checkpoint inhibitors (ICIs) has transformed the therapeutic landscape in cancer treatment (A. Ribas and JD Wolchok, Science, vol. 359, no. 6382, pp. 1350-1355, 2018), not all cancer types respond equally to ICIs, and even among those that do, only a portion of patients experience durable responses and favorable long-term outcomes. Intra- and inter-patient heterogeneity regarding immune responses may explain the variability in ICI efficiency. Therefore, to identify reliable predictive biomarkers that distinguish ICI responders from non-responders and to identify candidates for rational combination therapy, it is crucial to establish methods to analyze the interactions and behavior of single cells in real time in a non-artificial cellular environment, and such personalized approaches are now recognized as fundamental in the field of tumor immunology.

[0004] Several microscale tools exist to study single cell interactions. A common approach is to isolate a discrete number of cells in microwells, microchambers or droplets and monitor their interactions in multiple measurements (B. Dura and J. Voldman, Current opinion in immunology, vol. 35, pp. 23-9, 2015). Although these approaches have made it possible to elucidate the relationship between different immune responses (B. Dura and J. Voldman, Current opinion in immunology, vol. 35, pp. 23-9, 2015), they have some limitations due to the inability to investigate correlations between early signaling dynamics (calcium entry, formation of immune synapses...) and subsequent functional cellular events.

[0005] It is also important to be able to isolate live individual cells (e.g., individual T lymphocytes or cancer cells) after the interaction in order to analyze or modify the immunological state of the patient. Indeed, postsynaptic cell isolation is crucial for single-cell genomic analysis or for in vitro expansion, analysis, and adoptive transfer for in vivo studies.

[0006] Microfluidics is ideal for studying single cell behavior and isolating individual live cells post-synaptic. Some microfluidic devices can even study cellular interactions:

[0007] A device for pairing cells of different sizes has also been described by Fairuk A. Shaik et al, Pairing cells with different dimensions in a microfluidic device using a unidirectional flow, the 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences; 2020 October 4-9. The device consists of a microfluidic channel in which an array of trapping sites is arranged. Each trapping site is made of a three-layer structure consisting of a first layer that allows the cell-containing solution to flow along the microchannel from the bottom to the top of the microchannel, a second layer that forms a trap adapted to the expected size of a small cell, and a third layer that forms a trap adapted to the expected size of a large cell.

[0008] Another device for pairing cells is described by Shaik Faruk Azam et al, Pairing cells of different sizes in microfluidic device for immunological synapse monitoring. Lab Chip. 2022;22(5):908-20. The device consists of two parallel microfluidic channels separated by a wall with a synaptic opening and micropillars positioned on either side of the synaptic opening forming respective trapping sites adapted to hold a cell in the solution flowing through each channel. Summary of the Invention

[0009] However, in these devices, the trapping sites are of a predetermined fixed size and cannot efficiently pair cells of sizes different from the expected size. Moreover, once a cell is trapped in the trapping site, it is generally necessary to reverse the flow in the microchannel to release the cell from the trapping site, which is inconvenient.

[0010] As a result, despite the existence of highly complex systems, there currently exists no microfluidic device that i) allows for the analysis of spontaneous physical interactions and behavior of primary cells in a non-artificial environment, ii) allows for the immobilization of large numbers of cell pairs without affecting their viability, and iii) allows for the separation of cells after interaction so that they can potentially be expanded in vitro.

[0011] More precisely, there is currently no microfluidic cell pairing device that allows (i) screening of therapeutic or candidate molecules and (ii) studying the cell-cell interactions between live human primary lymphocytes and their partners (e.g. primary cancer cells) that allows the recovery of individual T cells or cancer cells after synapse formation. More precisely, no protocol exists to decipher the molecular events that occur at the scale of the immunological synapse between the patient's primary cancer cells and key T cells, both in the clinical context and in tumor dormancy.

[0012] The present invention provides an apparatus for trapping at least one cell pair in a solution comprising at least one first cell of a first type and at least one second cell of a second type, the apparatus comprising: - a microfluidic channel adapted for a unidirectional flow of said solution; - a first trap disposed within the microfluidic channel, the first trap comprising a pair of first fingers, at least one of the first fingers coupled to a respective first actuator configured to adjust the first trap along a direction transverse to the flow between an open position allowing passage of the first cell between the first fingers and a closed position conforming to a size of the first cell to allow trapping of the first cell between the first fingers; - a second trap disposed within the microfluidic channel, the second trap comprising a pair of second fingers, at least one of the second fingers coupled to a respective second actuator configured to adjust the second trap along a direction transverse to the flow between an open position allowing passage of the second cell between the second fingers and a closed position conforming to a size of the second cell to allow trapping of the second cell between the second fingers; Here, the first trap is positioned relative to the second trap to form a cell pair including the trapped first and second cells, such that the second cell has a physical or chemical interaction with the first cell when the first trap and the second trap are in a closed position.

[0013] In some embodiments, in the closed position, the second trap is larger than the first trap to allow trapping of a second cell larger than the first cell in the second trap.

[0014] In some embodiments, each first finger is coupled to a respective first actuator and each second finger is coupled to a respective second actuator.

[0015] The first and second actuators may be configured to move the first trap relative to the second trap to detach the first cell from the second cell and to open the first and second traps to release the trapped first and second cells into a stream.

[0016] In some embodiments, each first or second finger is coupled to a respective first or second actuator by a respective first or second rod substantially perpendicular to the flow.

[0017] Each of the first and second rods may support at least two first or second fingers, respectively, to form at least two first and second traps simultaneously adjustable by first and second actuators, respectively.

[0018] In some embodiments, the device may further comprise two pairs of foldable beams extending along the flow and connected to one of the first and second rods, each end of the foldable beams being fixed relative to the microfluidic channel, each foldable beam being deformable by a respective first or second actuator to move the respective first or second rod laterally relative to the flow to adjust the first or second trap.

[0019] In some embodiments, the device further comprises a control unit configured to receive sizes of the first and second cells and, based on the received sizes of the first and second cells, control the first and second actuators to adjust the distance between the first fingers and the distance between the second fingers, respectively, in closed positions of the first and second traps.

[0020] The apparatus may further comprise a measuring unit adapted to measure a size of the first and second cells in real time, and the control unit is configured to receive the measured sizes of the first and second cells in real time.

[0021] In some embodiments, the device further comprises at least one mechanical or electrical sensor adapted to detect trapping of a cell in each of the first, second and, where appropriate, third traps.

[0022] In some embodiments, at least one of the first actuators is configured to adjust a size of the first trap in a closed position after the first cell is trapped to enable trapping of a third cell in the first trap as well, forming a cell triplet with the second cell trapped in the second trap, and the first, second and third cells having a physical or chemical interaction.

[0023] In some embodiments, the device further comprises a third trap including a pair of third fingers disposed within the microfluidic channel, at least one of the third fingers coupled to a respective third actuator configured to adjust the third trap along a direction transverse to the flow between an open position allowing passage of the third cell between the third fingers and a closed position adapted to a size of the third cell to allow trapping of the third cell between the third fingers; Here, the third trap is positioned relative to the first and second traps such that when the first, second and third traps are in a closed position, the first, second and third cells physically or chemically interact to form a cell triplet comprising the trapped first, second and third cells.

[0024] In some embodiments, the device further comprises an array of electrically insulated electrodes disposed at the bottom of the microfluidic channel such that an overlap region of two electrodes is located beneath each trap, at least one surface of each electrode being exposed in a recess in the overlap region, and each electrode being connected to a power source to selectively apply a potential difference to the solution at each overlap region.

[0025] Another object of the present invention is a method for trapping 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, comprising: - flowing said solution through said microfluidic channel of said device; - actuating at least one first actuator to close the first trap, the size of the first trap in a closed position being adapted to the size of the first cell; - trapping a first cell in the first trap; - actuating at least one second actuator to close the second trap, the size of the second trap in a closed position being adapted to the size of the second cell; - trapping the second cell in the second trap, such that the second cell forms a cell pair with the first cell and the second cell has a physical or chemical interaction with the first cell.

[0026] Another object of the present invention is a method for trapping cell triplets in a solution comprising 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, comprising: - flowing a solution through said microfluidic channel of the device as described above; - actuating at least one first actuator to close the first trap, the size of the first trap in a closed position being adapted to the size of the first cell; - trapping a first cell in the first trap; - actuating at least one first actuator to adjust a size of the first trap in a closed position to match a size of both the first cell and the third cell; - trapping one third cell in the first trap; - actuating at least one second actuator to close the second trap, the size of the second trap in a closed position being adapted to the size of the second cell; - trapping a second cell in the second trap, wherein the second cell forms a triplet with the first and third cells, and the first, second and third cells have a physical or chemical interaction.

[0027] In some embodiments, the first and second traps are in an initial open position with the first trap separated from the second trap transversely to the flow, and after the first and second cells are trapped in the first and second traps, the first and / or second actuators are actuated to move the first and second traps closer together to form a cell pair or cell triplet.

[0028] The method may further comprise actuating the first and / or second actuator to move at least one of the first and second traps away from the other trap laterally relative to the flow, and opening at least one of the first and second traps to release cells trapped in the respective trap, thereby releasing at least one of the first, second and, if appropriate, third cells.

[0029] Alternatively, the method may comprise releasing at least one of the first, second and, where appropriate, third cells from a selected trap by applying a potential difference to electrodes underlying the trap that is greater than a potential difference causing electrolysis, dielectrophoresis or electroosmosis of the solution in the respective recess, thereby generating a gas bubble adapted to push the first, second and / or third cell out of the trap.

[0030] Another object of the present invention is a method for analyzing the real-time interaction of at least one pair or triplet of cells, comprising: - trapping at least one cell pair of the cell triplet in the manner described above; - acquiring data relating to an interaction of said cell pair or cell triplet with at least one of an optical sensor, an electrical sensor, a mechanical sensor, and a chemical sensor.

[0031] The method may further comprise exposing the at least one trapped cell pair or cell triplet to a solution having a determined pH and / or a determined viscosity, the pH or viscosity being selected to simulate cell interactions in a determined environment.

[0032] The present invention further provides a system for analyzing real-time interactions of at least one pair or triplet of cells, comprising: - the device as described above; and - at least one of an optical sensor, an electrical sensor, a mechanical sensor and a chemical sensor configured to obtain data regarding the interaction of pairs or triplets of cells trapped by said device.

[0033] Additional features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0034] [Figure 1] 1A-1E show the sequence of operation of a device according to an embodiment of the present invention to allow trapping of cell pairs; [Diagram 2] 2A-2F show the sequence of operation of a device according to one embodiment of the present invention to allow trapping of cell triplets; [Diagram 3] 3A-3C show a sequence of operation of an apparatus according to an embodiment of the invention to enable the release of a first type of cells, and FIG. 3D-3E show a sequence of operation of said apparatus to enable the release of a second type of cells; [Figure 4] 4A-4E show a sequence of an apparatus according to an embodiment of the present invention to allow simultaneous trapping of multiple cell pairs; [Diagram 5] FIG. 5 is a perspective view of an apparatus according to an embodiment of the present invention; [Figure 6]Figure 6 shows the trap of the device of Figure 5 in an open position; [Figure 7] FIG. 7 shows the trap of the device of FIG. 6 in a closed position. [Figure 8] Figure 8 shows the pairing of cells with the cell capture trap. a) SEM image shows a close-up of a trap with two capture sites. b) Bright field (top left) and fluorescence (top right) images of paired immune cells (white, Fura2-AM calcium probe) and leukemia cells (dark, DiI stained). Cells are paired regardless of size (bottom). [Figure 9] Figure 9 shows how cell activity can be monitored in cell pairs: Ca2+ imaging experiments showing activity (Ca2+ mobilization) of immune cells (natural killer cells, NK92 cells) after immune synapse (IS) formation with leukemia cells (K562 leukemia cells); [Figure 10] FIG. 10 shows a schematic of one embodiment of a device consisting of an array of electrodes configured to generate gas bubbles for selectively retrieving single cells, trapped cell pairs, or cell triplets from a trap; [Figure 11] 11A-11C show schematic diagrams of an embodiment of an electrode array. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] In this context, we propose herein a specific microfluidic cell pairing device set up to address these needs, i.e. to study cell-cell interactions between primary cells and their interacting partners (e.g., primary cancer cells and lymphocytes).

[0036] The device consists of a microfluidic channel that dynamically adjusts its shape to match the size of the patient's target cells and adapts to the unidirectional flow of solutions to trap cells of different sizes, such as lymphocytes and cancer cells paired together. This tunable cell-pairing device overcomes the limitations of current cell-pairing systems to manage differences in size and cell type.

[0037] By making the trapping structure mobile and actuated, we could adjust the size of the trapping zone in real time for different cell types and optimize the pairing of cells, so that different cell types are not stressed. Moreover, after pairing, the actuated trap can mechanically separate the cells and release them type by type. Finally, to increase the statistical validity of the assay, cells are preferably paired in parallel at multiple sites (on average about 100 cells).

[0038] As described below, the device allows for the analysis of molecular events occurring at the synapse between two cells, as well as molecular events occurring within the two cells during their interaction, for example by using real-time imaging or confocal microscopy. Furthermore, it is also possible to stimulate the trapped cells with candidate molecules and to identify specific biomarkers that may predict disease outcome or susceptibility to treatment.

[0039] Finally, and importantly, the device of the present invention allows individual cells to be harvested after synapse formation, grown in vitro for further analysis, and potentially returned to the patient after treatment.

[0040] The device of the invention includes a microfluidic channel adapted for unidirectional flow of a solution containing cells. The solution can consist of a mixture of at least two different types of cells. Alternatively, two solutions can flow sequentially through the microfluidic channel, one containing a first type of cells and one containing a second type of cells.

[0041] The first and second type of cells may be the same or different. In some embodiments, the first type of cells has a different size than the second type of cells; in the example developed below, the first type of cells has a smaller size than the second type of cells.

[0042] At least two adjustable traps are disposed in the microfluidic channel.

[0043] A first trap is configured to trap a first type of cell and a second trap is configured to trap a second type of cell. Typically, each trap is configured to trap one cell of each type. However, in some embodiments (see Figures 2A-2F, described in more detail below), one trap may be configured to trap two cells to form a cell triplet.

[0044] The traps may have different heights (height being the dimension perpendicular to the bottom of the microchannel), for example the larger the cell to be trapped the greater the height of the trap.

[0045] The traps do not have to be located at the same position in the microfluidic channel: in particular, if a first trap is intended to trap smaller cells than the second trap, the first trap may be located upstream of the second trap along the solution flow direction, the distance along said direction being selected to allow an interaction between the cells trapped in the first and second traps.

[0046] Each trap consists of 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 both fingers to adjust the first trap along a direction transverse to the flow between an open position allowing passage of a first cell between the fingers and a closed position adapted to the size of the cell to be trapped so that the cell can be trapped between the fingers. Having only one finger coupled to the actuator can simplify the design of the device. However, having both fingers coupled to respective actuators can increase the versatility of the device, for example allowing the entire trap to be displaced laterally. In particular, it may be advantageous to initially have the first and second traps offset from each other laterally to facilitate trapping of the first and second cells in the respective traps, and then to move the first and second traps laterally relative to each other to align them along the direction of the flow to form the cell pair.

[0047] As used herein, "trapping" means that the cell is held within the trap, preferably with minimal constraint on the cell by the fingers, so that the cell remains substantially free. To achieve this, the trap does not need to be completely closed, i.e., the fingers do not need to be in close contact with each other. Even in the closed position, the fingers are separated by a small gap, which is smaller than the size of a cell, preventing the cell from passing through the gap.

[0048] The first and second traps are positioned relative to one another such that, when the first and second traps are in a closed position, they form a cell pair consisting of the trapped first and second cells, within which an interaction between the first and second cells can occur.

[0049] 1A to 1E show schematic diagrams of the operation of the first and second traps as viewed from the top of a microfluidic channel.

[0050] 1A shows the first trap 1 with its fingers 10a, 10b and the second trap 2 with its fingers 20a, 20b in the open position. The direction of flow is indicated by the arrow F.

[0051] 1B shows that the fingers 10a, 10b of the first trap 1 are in a closed position, while the fingers 20a, 20b of the second trap 2 are still in an open position. To reach said closed position, the fingers 10a, 10b have been moved towards each other transversely to the flow F by respective actuators (not shown).

[0052] FIG. 1C shows the same configuration as FIG. 1B, with a first cell C1 trapped in the first trap 1.

[0053] 1D shows the first trap 1 in a closed position with a trapped first cell C1 and the second trap 2 in a closed position. To reach said closed position, the fingers 20a, 20b have been moved towards each other transversely to the flow F by their respective actuators (not shown).

[0054] FIG. 1E shows the same configuration as FIG. 1D, with a first cell C1 trapped in a first trap 1 and a second cell C2 trapped in a second trap 2. The first and second traps 1, 2 are close enough to each other to form a cell pair with the cells C1, C2, i.e., the interaction between the cells C1, C2 is permitted even if the cells C1, C2 are not in physical contact. One skilled in the art can design the size and arrangement of the traps depending on the size of the cells to be trapped and the type of interaction to be observed.

[0055] Of course, the invention is not limited to trapping a single cell pair, but can also be applied to trapping a cell triplet or a set of more cells, in which case the number of traps or the configuration of the traps can be adapted to construct the desired set of cells.

[0056] Trapping of cell triplets can be accomplished in a variety of ways.

[0057] 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 direction of flow. The size of the third trap is adjusted to the size of the cell to be trapped.

[0058] In another embodiment, as shown in Figures 2A-2F, one of the first and second traps (in this example, the first trap) is designed to trap two cells. In this way, the complexity of the device is increased to trap a third cell. There is no need to add a third trap that is different from the first and second traps.

[0059] 2A shows the fingers 10a, 10b of the first trap 1 in the closed position and the fingers 20a, 20b of the second trap 2 in the open position. The direction of flow is indicated by the arrow F.

[0060] FIG. 2B shows traps 1 and 2 in the same configuration as in FIG. 2A, with a first cell C1 trapped in the first trap 1.

[0061] Figure 2C represents the second trap 2 still in the open position. The first trap 1 has been partially reopened by moving finger 10b away from finger 10a while still holding the first cell C1. The opening width of the first trap 1 is adapted to allow trapping an additional cell C3, as shown in Figure 2D. Cell C3 may be of the same type as cell C1 or of a different type. Advantageously, cells C1 and C3 are substantially the same size.

[0062] FIG. 2E represents the first trap 1 in the same configuration as in FIG. 2D, with cells C1 and C3 trapped in trap 1 while the second trap 2 is in the closed position.

[0063] Figure 2F shows the first and second traps in the same configuration as in Figure 2E, with cell C2 trapped in the second trap 2. Thus, a cell triplet is formed by cells C1, C2, and C3.

[0064] This trapping device is particularly advantageous in that it not only allows the formation of cell pairs or cell triplets, but also allows the trapped cells to be easily released after the cell-cell interactions have been observed, without changing the flow in the microchannel. In contrast, for fixed traps, releasing trapped cells is very complicated and generally requires a counterflow of the flow F to force the cells out of the trap.

[0065] FIG. 3A shows traps 1 and 2 in the closed position with a pair of first and second cells C1 and C2 trapped in each trap.

[0066] As shown in Figure 3B, the second trap 2 moves away from the first trap 1 laterally with respect to the flow F, while the second trap 2 is still in the closed position. Thus, traps 1 and 2 are both closed but displaced from each other laterally. This displacement allows the first cell to be separated from the second cell.

[0067] As shown in Figure 3C, the first trap 1 is opened by moving at least one of the fingers 10a, 10b by its respective actuator, so that the cell C1 is released and leaves the trap 1 in the direction of flow F through the opening between the fingers 10a, 10b.

[0068] Figure 3D shows traps 1 and 2 in the closed position, with a pair of first and second cells C1, C2 trapped in each trap. The second trap 2 is displaced laterally away from the first trap 1 with respect to the flow F, while the second trap 2 is still in the closed position. Thus, traps 1 and 2 are both closed, but displaced laterally from each other. This displacement allows the first cell to be separated from the second cell.

[0069] As shown in Fig. 3E, the second trap 2 is opened by moving at least one of the fingers 20a, 20b by a respective actuator, so that 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 interfering with the first trap 1.

[0070] Of course, the sequences shown in Figures 3A-3C and 3D-3E can also be performed simultaneously, i.e., by moving the closed traps away from each other transversely to the flow and opening the first and second traps simultaneously.

[0071] As shown in Fig. 3E, the second trap 2 is opened by moving at least one of the fingers 20a, 20b by the respective 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. Because the first trap 1 is laterally offset from the second trap 2, the first trap does not prevent the cell C2 from flowing with the solution.

[0072] The device is then again ready for trapping a new cell pair.

[0073] Although Figures 1A through 3E illustrate the operation of the device to trap one cell pair or cell triplet, the device can of course also be designed to trap multiple cell pairs or triplets simultaneously.

[0074] For example, as shown in FIG. 4A, the device may be composed of a plurality of first traps 1 (e.g., five first traps) and a plurality of second traps 2 (e.g., five second traps). As described above, each first trap 1 is composed of fingers 10a and 10b that are movable relative to each other in a transverse direction to the flow F. Preferably, 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 is composed of fingers 20a and 20b that are movable relative to each other in a transverse direction to the flow F. Preferably, 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).

[0075] In Figure 4A, all the traps are in the open position. Preferably, each primary trap is aligned with a respective secondary trap in the direction of flow F.

[0076] As shown in Figure 4B, the first traps 1 are closed simultaneously. Preferably, both fingers of each first trap 1 are moved the same distance towards each other to keep the first trap aligned with the second trap.

[0077] As shown in FIG. 4C, a cell C1 is trapped in each of the first traps 1.

[0078] As shown in Figure 4D, the second trap 2 is closed at the same time. Preferably, both fingers of the second trap 2 are moved the same distance from each other to keep the second trap aligned with the first trap.

[0079] As shown in FIG. 4E, cell C2 is trapped in each of the first traps 2, thereby forming a cell pair with cell C1 trapped in each of the first traps 1.

[0080] Once a cell pair is observed, the first cell can be released simultaneously and the second cell can be released simultaneously by operating the device as described with reference to Figures 3A-3E, or the entire cell pair can be released simultaneously by opening all the traps.

[0081] The actuator is controlled by a control unit.

[0082] The control unit is configured to receive sizes of the first and second cells and control the first and second actuators to adjust, in a closed position of the first and second traps, a gap between the fingers of the first trap and a gap between the fingers of the second trap, respectively, based on the received sizes of the first and second cells, wherein the gaps are smaller than the respective cells so as to retain the cells within the traps.

[0083] In some embodiments, the sizes of the first and second cells may be manually entered by a user, for example via a user interface coupled to the control unit.

[0084] In other embodiments, the device can include a measurement unit coupled to the control unit and adapted to measure the size of the first and second cells in the flow of solution in real time, such that the control unit can receive the measured sizes of the first and second cells in real time and adjust the operation of the actuator accordingly to adapt the gap between the fingers in the closed position.

[0085] In certain embodiments, the device can include at least one mechanical or electrical sensor adapted to detect when a cell is trapped within the trap.

[0086] For example, the mechanical sensor can specifically detect the mechanical force exerted by the cells 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 frequency shift of the actuator. Mechanical sensing can also be performed by static measurements, where one actuator can be displaced to compress the cells. The displacement induced in the second actuator by the stiffness of the cells is due to the presence of the cells. Thus, the number of cells can be assessed by the displacement of the second actuator.

[0087] An electrical sensor can detect the current flowing between the fingers of the trap through the cells. Electrical sensing can be accomplished by measuring the electrical conductance between pairs of actuators with and without trapped cells, and determining the number of trapped cells based on the decrease in conductance due to the fact that cells are less electrically conductive than the solution.

[0088] The device can be used in a system for analyzing the interaction of at least one pair or triplet of cells in real time.

[0089] The system can include optical, electrical, mechanical, and / or chemical sensors configured to obtain data related to interactions of pairs or triplets of cells trapped by the device. The electrical and mechanical sensors can be used to confirm the presence of cells as described above, as well as to determine the number of trapped cells and measure mechanical and / or electrical properties of the trapped cells. The optical and chemical sensors can detect fluorescence to image the pairing of cells and detect and quantify cellular interactions, such as activation of an immune response between these cells.

[0090] FIG. 5 is a perspective view of an apparatus according to an embodiment of the present invention.

[0091] The device consists of a microfluidic channel 3, the direction of flow of the cell-containing solution being indicated by the arrow F.

[0092] As can be seen best in Figures 6 and 7, the device consists of two rows of traps, each row consisting of three first traps 1 and three second traps 2. Figure 6 shows the traps in the open position and Figure 7 shows the traps in the closed position.

[0093] As explained above, each first trap 1 consists of a finger 10a and a finger 10b, and each second trap 2 consists of a finger 20a and a finger 20b.

[0094] The device is composed of four actuators 11a, 11b, 21a, 21b arranged on either side of the trap. Each actuator can be a programmable stepper motor. For example, the actuators can be piezoelectric linear motors.

[0095] Actuator 11a is configured to move fingers 10a of each first trap, actuator 11b is configured to move fingers 10b of each first trap 1, actuator 21a is configured to move fingers 20a of each second trap, and actuator 21b is configured to move fingers 20b of each second trap.

[0096] To that end, each finger 10a, 10b, 20a, 20b is coupled to a respective actuator 20a, 20b, 21a, 21b by a respective rod 12a, 12b, 22a, 22b. The rods 12a, 12b, 22a, 22b extend substantially perpendicular to the flow F.

[0097] Thus, for each row of traps, the three first trap 1 fingers 10a are supported by rods 12a and can be moved simultaneously by actuators 11a, the three first trap 1 fingers 10b are supported by rods 12b and can be moved simultaneously by actuators 11b, and similarly, the three second trap 2 fingers 20a are supported by rods 22a and can be moved simultaneously by actuators 21a, and the three second trap 2 fingers 20b are supported by rods 22a and can be moved simultaneously by actuators 21b.

[0098] Advantageously, the device consists of 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 a respective actuator 11a, 11b, 21a, 21b. The rods extend in a direction substantially perpendicular to the beams.

[0099] Each end of the beam is fixed relative to the microchannel, but each beam is deformable laterally to the flow F by a respective actuator 11a, 11b, 21a or 21b to move a respective rod 12a, 12b, 22a or 22b laterally to the flow F to adjust the first or second trap.

[0100] To minimize assembly, each beam can be integrally formed with its corresponding rod and fingers. For example, the beams, rods and fingers can be made from a photopolymerizable resin using methods known in the microelectronics art.

[0101] Of course, the invention is not limited to the illustrated embodiment, and the actuators and the mechanical transmission between the actuators and the fingers can be implemented in different embodiments, and the number of traps can be adjusted according to the experimental needs.

[0102] In some embodiments, the release of a selected trapped single cell or trapped cell aggregate (cell pair or cell triplet) is achieved by the generation of gas bubbles by electrolysis of the solution surrounding the trap. To that end, the device includes a plurality of electrically insulated electrodes arranged to form an array at the bottom of a microchannel. A node of the array (i.e., an area where two electrodes overlap) is located below the trap. In this way, when a sufficient potential difference (i.e., a potential difference larger than that which induces electrolysis of the solution) is applied between the two overlapping electrodes, a gas bubble is generated at the bottom of the microfluidic channel, which rises substantially vertically through the solution and pushes the cell or cell aggregate out of the trap. The released cell or cell aggregate is driven along the microfluidic channel by the flow of the solution.

[0103] FIG. 10 shows a schematic of an array of electrodes arranged at the bottom 30 of a microfluidic channel. A first set of electrodes E11, E12, E13 extend perpendicular to the direction of flow F, while a second set of electrodes E21, E22, E23 extend parallel to the direction of flow F and thus perpendicular to the first set of electrodes E11, E12, E13. Each electrode of the first set intersects with an electrode of the second set below its respective trap. By "below" we mean that the intersection or overlap region is aligned with the trapping zone along a line perpendicular to the bottom of the microfluidic channel.

[0104] The first and second electrode sets are electrically insulated from each other.

[0105] Each electrode is connected to an electrical source configured to selectively apply a determined potential to each electrode, resulting in a potential difference across each intersection.

[0106] Depending on the potentials applied to each electrode, the potential difference may be greater than a potential threshold that allows electrolysis of the solution flowing through the microfluidic channel. In such a case, gas bubbles are generated at the intersection region, rising through the solution and pushing the cells or cell aggregates out of their respective traps. Thus, the cells or cell aggregates can be removed from the traps without actuating actuators to move the fingers apart.

[0107] Conversely, if the potential difference is below the potential threshold, electrolysis does not occur and the cell aggregates remain within the trap.

[0108] For example, in the embodiment shown in Figure 10, a potential of 3V is applied to electrodes E11, E13, E21, E23, a potential of 1V is applied to electrode E12, and a potential of 5V is applied to electrode E22. As a result, a potential difference ΔP1 = 0V is applied to the intersections of electrodes E13 and E21 and between electrodes E13 and E23, a potential difference ΔP2 = 2V is applied to the intersections of electrodes E12 and E21 and between electrodes E11 and E22, and a potential difference ΔP3 = 4V is applied to the intersections of electrodes E12 and E22. Of course, the number of electrodes in each set and their relative arrangement are presented for illustrative purposes only and are not intended to be limiting.

[0109] Assuming that a potential difference of at least 3 V is required for electrolysis of the solution, the potential differences ΔP1 and ΔP2 are too low for electrolysis to occur at the corresponding intersections, but electrolysis does occur at the intersection between electrodes E12 and E22 because ΔP3 is greater than 3 V. As a result, a gas bubble B forms at the intersection and rises substantially vertically, pushing the cell pair C1, C2 out of the trap.

[0110] The applied potential difference depends on the electrode material, electrode geometry, and the ionic strength of the solution (buffer or medium) and can be up to 20 VDC. AC signals (up to 1 or 2 MHz) can also be applied for dielectrophoresis or AC electroosmosis to release cells, cell pairs, or cell triplets.

[0111] The electrode array can be fabricated by patterning a conductive material (e.g., indium tin oxide (ITO), metal such as gold, etc.) to form a first set of electrodes (e.g., a set of parallel electrodes) on the bottom surface of a microfluidic channel. The thickness of the electrodes can reach up to 500 nm. These electrodes are covered with a first dielectric layer, whose thickness is greater than that of the electrodes, electrically insulating each electrode. The dielectric material can be, for example, SiO2, spin-on glass, or Cytop, a fluororesin. TM It is.

[0112] The second set of electrodes is patterned perpendicular to the first set of electrodes and covered with a second dielectric layer with a thickness greater than that of the electrodes to electrically insulate each electrode. The width of each electrode is on the same order as the size of the cell to be captured, e.g., a few micrometers.

[0113] The dielectric layer is then etched around the overlapping electrode regions to form recesses that expose at least a portion of each electrode to solutions flowing through the microfluidic device. Figures 11A-11C show various embodiments of such recesses in schematic form.

[0114] FIG. 11A shows a portion of the bottom of a microfluidic device according to an embodiment, which is composed of a portion of a first electrode E11 and a portion of a second electrode E21 partially overlapping the first electrode. As described above, the electrodes E11 and E21 are embedded in a first and second dielectric layer (represented here as a single dielectric layer 31). A recess 300 is formed through the dielectric layer 31 to the bottom 30 of the microfluidic channel. As best seen in FIG. 11B, which is a partial cross-sectional view of the recess of FIG. 11A, the etching partially exposes the top and side surfaces of the second electrode E21 on either side of the overlapping second electrode E21 as well as the top and side surfaces of the first electrode E11. However, the etching does not remove the dielectric material located between the first and second electrodes.

[0115] The recesses may have a circular shape as depicted in Figures 11A-11B, but may have any suitable shape, for example a rectangle or a square (as shown in Figure 11C). In the illustrated embodiment, the recesses are in the center of the overlapping regions, but different arrangements are possible, provided that at least a portion of the top and / or side surfaces of each electrode are exposed.

[0116] FIG. 11C shows a part of the bottom of a microfluidic device according to another embodiment, consisting of a part of a first electrode E11 and a part of a second electrode E21 partially overlapping the first electrode. As in FIG. 11A, the electrodes E11 and E21 are embedded in a first and a second dielectric layer (here represented as a single dielectric layer 31). A recess 300 is formed through the dielectric layer 31 to the bottom 30 of the microfluidic channel. Contrary to the embodiment of FIG. 11A, the etching exposes only the sides of the first and second electrodes E11, E21. In this embodiment, the recess is thus formed along one side of the electrodes E11, E21. As mentioned above, the etching does not remove the dielectric material located between the first and second electrodes.

[0117] The recesses allow the solution to contact the electrodes only at or near the overlapping portions, and each recess is formed directly beneath a trap where cells can aggregate.

[0118] The size of the recess (e.g., diameter for circular recesses, or length / width for square or rectangular recesses) is selected to generate a bubble of suitable size for extruding cells or cell aggregates. To that end, the size of the recess (and resulting bubble) is equal to or slightly larger than the size of the cell, which may be on the order of 10 μm.

[0119] Moreover, the device of the present invention can be used not only to study interactions between immune cells as described above, but also to study physical or chemical interactions between any kind of cells, i.e. cells of different sizes, cells of different sources, cells of cell lines or primary cells, naturally occurring cells or cells produced recombinantly. In particular, these cells can be circulating cells, such as blood cells (red blood cells, platelets, granulocytes, agranulocytes, etc.), urine cells, sperm cells, adipocytes (white or brown adipocytes), metastatic or circulating tumor cells, or tissue-associated cells, such as epithelial cells, fibroblasts, oocytes, solid cancer cells, skin cells (keratinocytes, melanocytes, Merkel cells, Langerhans cells), muscle cells (skeletal muscle cells, cardiac muscle cells, smooth muscle cells), nerve cells (neurons, glial cells, etc.), endothelial cells, pancreatic cells, chondrocytes, bone cells (osteoblasts, osteoclasts, osteocytes, etc.). They can be differentiated cells or stem cells. They can be prokaryotic cells (bacteria, etc.) or eukaryotic cells (including yeast cells).

[0120] In order to be properly trapped in the device of the invention, the cells to be studied preferably have an average diameter greater than about 0.5 μm, however, in certain embodiments it is possible to use the device of the invention to analyze the behavior of these cells when in contact with smaller microorganisms such as bacteria, viruses, protozoa, etc.

[0121] The device of the present invention can tolerate some cell clumping. However, in certain embodiments, the concentration of cells in the sample to be analyzed should be adjusted so that there is little or no cell clumping. For example, the cell concentration in the solution run through the device of the present invention is preferably 500,000 cells / ml or less. Higher concentrations can be diluted with the medium required for the survival of the cell type.

[0122] It is advisable to isolate cells from the blood sample before inserting it into the device. As an example, the blast cells used in the following experiments were isolated from peripheral blood after Ficoll separation and lymphocyte depletion using immunomagnetic negative selection (CD3 for T lymphocytes and CD20 for B lymphocytes).

[0123] In particular, solutions used as flow channels (such as culture media and other solutions, e.g., plasma) can be filtered so as to be free of cells or other components larger than 2 μm in diameter (if the cells being studied have a diameter larger than 5 μm), or larger than one-third the diameter of the cells being studied (if the cells have a diameter smaller than 5 μm).

[0124] Once the target cells are trapped within the device of the present invention, many imaging techniques can be used to analyze the behavior of the cells. Imaging methods are the most powerful tools to visualize the structure of cells and their live behavior. Many microscope-based imaging approaches have been developed so far, including confocal microscopes, super-high resolution microscopes, two-photon microscopes, electron microscopes, and atomic force microscopes. In imaging, proteins, RNAs, lipids, and glycans can be quantified using labeled antibodies.

[0125] Analysis of real-time interactions of trapped cell pairs or triplets can include exposing the cells to solutions with specific properties, such as pH or viscosity, to simulate cell interactions under specific conditions. One or more solutions with different properties can be flowed through the microfluidic channel to selectively access specific cells or to access the entire trapped cell pair or triplet collectively.

[0126] In some embodiments, selected cells, or entire trapped cell pairs or triplets, can be exposed to solutions of varying pH, particularly solutions with a lower acidic pH (e.g., about 6.2-6.5) than that found in normal tissues (about 7.4), allowing for the variability present in vivo or in non-healthy conditions (such as the acidic microenvironment of cancer tissue or bone marrow).

[0127] In other embodiments, selected cells, or entire trapped cell pairs or triplets, can be exposed to solutions having various viscosities, for example to mimic cellular interactions in blood (the solutions thus have a viscosity of 3-5 cP (3-5 mPa.s), corresponding to the average blood viscosity) and / or to mimic cellular interactions in bone marrow (the solutions thus have a viscosity of 30-38 mPa.s, corresponding to the average bone marrow viscosity).

[0128] 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 altered by the cell-cell interaction. For example, flow cytometry can quantitatively and high-throughput analyze individual cells. Multiplex labeling identifies the composition of various cell types based on the expression of surface protein markers. In addition, flow cytometry can quantify multiple proteins in single cells using dyes or fluorescently labeled antibodies. Similarly, flow cytometry has also been used to measure mRNA or microRNA expression levels using labeled antisense nucleotides. When combined with Fluorescence Activated Cell Sorting (FACS), target cells can be separated and recovered in a high-throughput manner, allowing for more comprehensive gene expression and proteomic analysis. Imaging flow cytometry is also a high-throughput approach that analyzes thousands of individual cells per second by simultaneously taking images of the cells and providing morphological information. Recent advances in droplet-based technology have enabled transcriptome profiling of single cells. When combined with DNA barcoding, thousands of cells can now be analyzed. Because transcription and actual protein abundance often differ, proteome analysis in single cells is needed. Although comprehensive analysis of proteins is not yet possible, mass cytometry is one of the most high-throughput protein analyses. Isolated cells are labeled with multi-metal-conjugated antibodies and the amount of target proteins is detected by a Cytometry by Time of Flight (CyTOF) mass spectrometer. Current technology allows the measurement of about 40 different proteins simultaneously. Imaging-based Mass Spectrometry (IMS) is an approach that integrates the strengths of imaging by matrix-assisted laser desorption / ionization with unbiased proteome analysis. By measuring the mass / charge signal at various coordinates, both the presence of target molecules and information on their localization in tissues can be analyzed.IMS allows the analysis of thousands of proteins, lipids, and metabolites without labeling directly from tissue. For other molecular tools, see Nishida-Aoki N. and Gujral TS, Emerging approaches to study cell-cell interactions in tumor microenvironment, Oncotarget, 2019, 10(7):785-797.

[0129] In a preferred embodiment, the device of the invention is used to study the Immunological Synapse (IS), i.e. the cell pair involving an immune cell and its target cell. Functional analysis at the level of the Immunological Synapse (IS) is fundamental to improve the efficiency of immunotherapies currently used in the clinic. Indeed, despite very promising results shown with immunotherapies targeting advanced cancers, the majority of patients do not respond to certain treatments. In this context, it is important to identify relevant biomarkers that could predict the efficiency of these therapeutic molecules.

[0130] The immunological synapse consists of antigen-presenting cells (e.g., B cells, dendritic cells, macrophages, virus-infected cells, and cancer cells) and CD4 + T lymphocytes, CD8 + Target cells such as T lymphocytes, B lymphocytes, natural killer cells (NK cells) are involved. In particular, the device of the present invention can be used to study specific interactions between dendritic cells and T cells, or between tumor cells and T cells.

[0131] In a particularly preferred embodiment, the device of the present 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 immune synapse responses in the clinical context of cancer. The device of the present invention allows the study of the immune synapse between T cells and cancer cells, the restoration of T lymphocyte activity, or understanding the molecular mechanisms underlying resistance to therapeutic molecules, making it an extremely valuable tool in the development of precision medicine.

[0132] Although T lymphocytes are generally involved in the elimination of tumor cells, inhibitory signaling pathways targeted at immune effector cells and tumor cells may 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). Furthermore, it has been proposed that tumor cells exhibit characteristics (quiescence, dormancy, stemness...) that lead to resistance to T lymphocytes (B. Quesnel, Acta Pathologica, Microbiologica, et Immunologica Scandinavica, vol. 116, no. 7-8, pp. 685-94, 2008;). Thus, understanding the dialogue between T cells and tumor cells (Y. Touil et al., Scientific reports, vol. 6, pp. 30405, 2016) is crucial to prevent relapse in patients.

[0133] Thanks to the device of the invention, the interaction between T cells and tumor cells can be monitored by various conventional means (e.g. transcriptomics, proteomics, genomics) and more precisely by, for example, confocal microscopy, Western blot and / or flow cytometry, as explained above, to determine the level of Ca. 2+ This can be observed by checking the signaling (see Figure 8), the expression of checkpoint inhibitor proteins, or stem markers.

[0134] The preferred analytical tool is calcium signaling. It is well known that calcium signals govern T cell activity (M. Trebak and JP Kinet, Nature reviews Immunology, vol. 19, no.3, pp. 154-169, 2019). Moreover, calcium signaling is associated with all hallmarks of cancer (N. Prevarskaya, R. Skryma, and Y. Shuba, Physiological reviews, vol. 98, no. 2, pp. 559-621, 2018).

[0135] The device of the present invention is suitable for performing functional studies on the immunological synapses formed with different sized primary cells from patients, despite limited access to rare but important dysfunctional T cells or dormant tumor cells from the patient.

[0136] Thus, in a preferred embodiment, the device of the present invention allows deciphering the calcium signature of the immunological synapse in the dormant state of the tumor.

[0137] In a particular aspect, the present invention proposes: - A system that can simultaneously analyze the functions of two types of cells (T cells and tumor cells from the same patient) during immunological synapse formation and track subsequent cellular events. - A system that allows to contact rare cells at low frequencies and perform functional studies. This allows to analyze immunological synapse formation, an event that is rare in vivo. Indeed, very low numbers of cells are needed to perform experiments with the device of the invention. - a system that allows i) to reactivate T lymphocytes and evaluate their efficiency against tumor cells of different stages or ii) to screen for therapeutic or candidate molecules capable of sensitizing resistant cancer cells to T cells.

[0138] These tests can be performed clinically to evaluate the activity of T lymphocytes against tumor cells (calcium response, lysis) at key stages of the disease (diagnosis, dormant or minimal residual disease, relapse).

[0139] Ca associated with IS formation 2+ Signal transduction and the resulting cellular events are driven by Ca 2+ Ca can be assessed by imaging and confocal microscopy. 2+ Signal transduction can be investigated using specific inhibitors, agonists, antagonists, anti-inhibitory checkpoints (antibodies), therapeutic or candidate molecules. Elucidation of such control mechanisms may provide new perspectives for immunotherapy or help optimize current immunotherapy protocols.

[0140] Once the cells are released from the trap, they can be further analyzed. It is then advantageous to specifically collect cells of the same category (e.g., only T cells or only tumor cells) by sorting with flow cytometry. Conventional molecular analyses (immunostaining, Western blotting, PCR, spectrometry, proteomics, etc.) can then be performed to identify specific genomic or proteomic signatures or biomarkers in these cells.

[0141] By studying the immunological synapse and then analyzing the released T cells and resistant tumor cells, it may be possible to obtain genomic signatures specific to each stage of cancer disease or each patient. In such cases, the device of the present invention allows the discovery of new biomarkers predicting disease outcome. EXAMPLES

[0142] Prior to cell loading, isolated NK cells and leukemia K562 cells were cultured for 24 h to remove the staining antibodies used for phenotypic sorting. Cancer cells were stained with a viability / tracker dye (DiI). Single-cell Ca at the IS 2+Signaling was monitored using SEM (see Figures 8 and 9). 2+ The sensitive Fura-2 dye detects cytoplasmic Ca 2+ Changes in the concentration of basal Ca can be monitored in real time. 2+ The signal (without stimulation) is compared to that observed with NK cells.

[0143] This protocol aims to measure intracellular Ca during IS formation. 2+ It can evaluate level fluctuations, but can also be used to monitor: - lysis due to cytotoxicity, - resistance to dissolution, - The effect of IFN-g and TNF-α (cytokines) release in the IS on calcium responses; - others

[0144] Alternatively, we used T cells instead of NK cells and HLA-DR preincubated with different concentrations of superantigen. + The IS can also be activated using cancer cells or by pre-coating with different concentrations of anti-CD3 antibodies. The cells are then loaded with ratiometric Fura-2 dye and Ca 2+ Signal studies can be performed.

[0145] In particular, Ca in IS 2+ The expression and localization of key proteins involved in signal transduction, cytotoxic activity, apoptosis, proliferation, and cytokine (IFN-g, TNF-α) release can be assessed. 2+ The molecular nature of the channels involved in the reaction can be determined using selective inhibitors that are added directly to the microfluidic system. By modulating calcium signaling of the IS with selective agonists, antagonists, therapeutic or candidate molecules, the function of T lymphocytes (proliferation, cytokine secretion, cytotoxic lysis) and the possibility of restoring the proliferation / quiescence of cancer cells can be evaluated.

Claims

1. An apparatus for trapping at least one cell pair in a solution comprising at least one first cell (C1) of a first type and at least one second cell (C2) of a second type, - Microfluidic channels (3) adapted to the unidirectional flow (F) of the solution; - A first trap (1) disposed within the microfluidic channel (3), comprising a pair of first fingers (10a, 10b), wherein at least one of the first fingers (10a, 10b) is coupled to a first actuator (11a, 11b), the first actuator being configured to adjust the first trap (1) laterally with respect to the flow (F) between an open position that allows the passage of the first cell between the first fingers (10a, 10b) and a closed position that conforms to the size of the first cell to allow trapping of the first cell between the first fingers (10a, 10b); - A second trap (2) disposed within the microfluidic channel (3), comprising a pair of second fingers (20a, 20b), wherein at least one of the second fingers (20a, 20b) is coupled to a second actuator (21a, 21b), the second actuator being configured to adjust the second trap (2) laterally with respect to the flow (F) between an open position that allows the passage of the second cell between the second fingers (20a, 20b) and a closed position that conforms to the size of the second cell to allow trapping of the second cell between the second fingers (20a, 20b); Equipped with, The first trap (1) is positioned relative to the second trap (2) such that when the first trap and the second trap are in the closed position, the second cell forms a cell pair including the trapped first and second cells so that the second cell interacts physically or chemically with the first cell. Device.

2. The apparatus according to claim 1, wherein, in the closed position, the second trap (2) is larger than the first trap (1) to allow trapping of a second cell larger than the first cell within the second trap.

3. The apparatus according to claim 1 or 2, wherein each first finger (10a, 10b) is coupled to each first actuator (11a, 11b), and each second finger (20a, 20b) is coupled to each second actuator (21a, 21b).

4. The apparatus according to claim 3, wherein the first and second actuators (11a, 11b, 21a, 21b) are configured to move the first trap relative to the second trap to detach the first cells from the second cells, and to open the first and second traps to release the trapped first and second cells into the flow (F).

5. The apparatus according to claim 1, wherein each first or second finger (10a, 10b, 20a, 20b) is coupled to each first or second actuator (11a, 11b, 21a, 21b) by each first or second rod (12a, 12b, 22a, 22b) substantially perpendicular to the flow (F).

6. The apparatus according to claim 5, wherein each of the first and second rods (12a, 12b, 22a, 22b) supports at least two first or second fingers (10a, 10a, 10b, 10b', 20a, 20a', 20b, 20b') such that each of the first and second rods (12a, 12b, 22a, 22b) is simultaneously adjustable by the first and second actuators (11a, 11b, 21a, 21b).

7. The apparatus according to claim 5, further comprising two pairs of foldable beams (13a, 13b, 23a, 23b) extending along a flow (F) and connected to one of first and second rods (12a, 12b, 22a, 22b), wherein each end of the foldable beams is fixed to the microfluidic channel (3), and each foldable beam is deformable by the respective first or second actuators (11a, 11b, 21a, 21b), and the first or second trap is adjusted by moving the respective first or second rods laterally with respect to the flow (F).

8. The apparatus according to claim 1, further comprising a control unit configured to receive the sizes of the first and second cells and, based on the received sizes of the first and second cells, to control the first and second actuators (20a, 20b, 21a, 21b) to adjust the distance between the first fingers and the distance between the second fingers in the closed position of the first and second traps, respectively.

9. The apparatus according to claim 8, further comprising a measuring unit adapted to measure the sizes of the first and second cells in real time, wherein the control unit is configured to receive the measured sizes of the first and second cells in real time.

10. The apparatus according to claim 1, further comprising at least one mechanical or electrical sensor adapted to detect that a cell has been trapped in a first, second, and, where appropriate, third trap.

11. The apparatus according to claim 1, wherein at least one of the first actuators is configured to adjust the size of the first trap in a closed position after the first cell has been trapped, thereby allowing one third cell to also be trapped within the first trap, forming a cell triplet with the second cell trapped within the second trap, and the first, second, and third cells to interact physically or chemically.

12. The microfluidic channel (3) further comprises a third trap including a pair of third fingers, at least one of the third fingers being coupled to a third actuator, the third actuator being configured to adjust the third trap lateral to the flow (F) between an open position that allows the passage of the third cell between the third fingers and a closed position adapted to the size of the third cell to allow trapping the third cell between the third fingers. The apparatus according to claim 1, wherein the third trap is positioned relative to the first and second traps such that when the first, second and third traps are in the closed position, the first, second and third cells interact physically or chemically to form a cell triplet comprising the trapped first, second and third cells.

13. The apparatus according to claim 1, further comprising an array of electrically insulated electrodes (E11, E12, E13, E21, E22, E23) positioned at the bottom (30) of the microfluidic channel such that the overlapping regions of two electrodes are located beneath each trap, at least one surface of each electrode is exposed in the recess (300) of the overlapping region, and each electrode is connected to a power source to selectively apply a potential difference to the solution in each overlapping region.

14. A method for trapping at least one cell pair in a solution comprising at least one first cell of a first type and at least one second cell of a second type, comprising the following steps: - The solution is flowed through the microfluidic channel of the apparatus according to claim 1; - At least one first actuator is activated to close the first trap, and the size of the first trap in the closed position is adapted to the size of the first cell; - Trapping one first cell in the first trap; - At least one second actuator is activated to close the second trap, and the size of the second trap in the closed position is adapted to the size of the second cell; - The second cell is trapped in the second trap so that the second cell forms a cell pair with the first cell, and the second cell interacts with the first cell physically or chemically.

15. A method for trapping a cell triplet in solution comprising 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 the following steps: - The solution is flowed through the microfluidic channel of the apparatus according to claim 1; - At least one first actuator is activated to close the first trap, and the size of the first trap in the closed position is adapted to the size of the first cell; - Trapping one first cell in the first trap; - Activate at least one first actuator to adjust the size of the first trap in the closed position to fit the size of both the first and third cells; - Trapping one third cell in the first trap; - At least one second actuator is activated to close the second trap, and the size of the second trap in the closed position is adapted to the size of the second cell; - A second cell is trapped in the second trap, so that the second cell forms a triplet with the first and third cells, and the first, second and third cells interact physically or chemically.

16. The method of claim 14, carried out using the apparatus of claim 5, wherein the first and second traps are initially in an open position, with the first trap separated from the second trap in a direction lateral to the flow (F), and after the first and second cells are trapped in the first and second traps, the first and / or second actuators are activated to bring the first and second traps closer together to form a cell pair or cell triplet.

17. The method according to claim 16, further comprising activating the first and / or second actuators to move at least one of the first and second traps laterally with respect to the flow (F) away from the other trap, opening at least one of the first and second traps to release the cells trapped in each trap, thereby releasing at least one of the first, second, and optionally third cells.

18. The method of claim 14, carried out using the apparatus of claim 13, wherein a potential difference greater than the potential difference that causes electrolysis, dielectrophoresis, or electroosmosis of the solution in each recess (300) is applied from the selected trap to electrodes (E11, E21) overlapping below the trap, thereby generating bubbles (B) adapted to push the first, second, and / or third cells out of the trap, and releasing at least one of the first, second, and optionally third cells.

19. A method for analyzing real-time interactions of at least one pair or triplet of cells, comprising the following steps: - Trapping at least one cell pair of a cell triplet by the method of claim 14; - Data is obtained regarding the interaction between the cell pair or cell triplet and at least one of the optical sensor, electrical sensor, mechanical sensor, or chemical sensor.

20. The method according to claim 19, further comprising exposing the at least one trapped cell pair or cell triplet to a solution having a determined pH and / or a determined viscosity, wherein the pH or viscosity is selected to simulate cell interactions in a determined situation.

21. A system for analyzing real-time interactions of at least one pair or triplet of cells, comprising the following: - The apparatus according to claim 1; and - At least one of optical, electrical, mechanical, and chemical sensors configured to acquire data relating to the interaction of a pair or triplet of cells trapped by the apparatus.

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