Microfluidic devices and methods for forming cell aggregates, and methods for selectively processing cells within cell aggregates.

Abstract

The present invention relates to a microfluidic device (1) for forming a cell aggregate comprising at least one first cell (C1) and one second cell (C2), and for individually processing selected cells of the cell aggregate: - Microfluidic channel (10); - At least one main inlet (11) for fluids containing a first cell, a second cell, and a third cell, respectively, located in the first portion (101) of the microfluidic channel; - An outlet (12) located in the second portion (102) of the microfluidic channel for controlling the flow rate of fluid within the microfluidic channel; - A first auxiliary inlet (131) for at least one first auxiliary fluid, located in the first portion (101) of the microfluidic channel upstream or downstream of the main inlet (11); - At least one cell trapping section (14) positioned between the first and second parts within the microfluidic channel; - At least one first valve to control the flow rate of the first auxiliary fluid, causing the fluid containing the first cells and the second cells to flow at a predetermined height within the microfluidic channel, thereby guiding the first cells and the second cells to the first and second capture units, respectively. Equipped with, The microfluidic device relates to a microfluidic device in which each cell capture section (14) comprises at least one first capture section (141) and one second capture section (142), each first and second capture section being sized to accommodate a first or second cell, the first and second capture sections being adjacent to each other in a direction perpendicular to the bottom (100) of the microfluidic channel, and forming a cell aggregate containing the captured first and second cells, each cell being at a different height relative to the bottom of the microfluidic channel.

Description

[Technical Field]

[0001] The present invention relates to a microfluidic device and method for forming cell aggregates, and to a method for selectively processing cells within a cell aggregate. [Background technology]

[0002] Cell-cell interactions play a crucial role in various biological systems, and cell pairing is particularly important in immunity, where it is involved in the induction and mediation of many important developmental immune responses (selection, proliferation, differentiation) and functional immune responses (cytolysis, cytokine and antibody production). In this context, a deeper understanding of the interaction dynamics between immune cells and their cellular partners is essential.

[0003] Various microfluidic devices are described that enable the capture of cells and the formation of cell aggregates such as cell pairs and cell triplets.

[0004] In particular, FA Shaik et al. [1] disclose a microfluidic device, as shown in Figure 1, comprising a microfluidic channel 10', a fluid inlet 11' containing cells located in a first portion 101' of the microfluidic channel, an outlet 12' located in a second portion 102' of the microfluidic channel to control the flow rate of fluid within the microfluidic channel, and a capture section 14' located between the first and second portions to capture cells. The capture section comprises a first capture section 141' and second capture sections 141, 142'. The first lower capture section 141' has a width and height that can hold small cells relative to the bottom 100' of the microfluidic channel, and the second upper capture section 142' has a width and height that can hold larger cells relative to the bottom 100' of the microfluidic channel. In the first step (i), a solution containing small cells C1 is introduced into the microfluidic channel through a first inlet (left side of the figure, not shown), and an auxiliary fluid is introduced into the microfluidic channel through a second inlet 13', causing the solution to flow along the bottom 100' of the microfluidic channel. As a result, the small cells can be captured in the first lower capture section. In the second step (ii), the flow rate of the auxiliary fluid is increased to withdraw the solution containing the small cells. In the third step (iii), a solution containing larger cells C2 is introduced into the microfluidic channel through the first inlet. In this step, the second inlet is closed, and no auxiliary fluid flows into the microfluidic channel. In this way, the larger cells can be captured in the second upper capture section 142' and brought into contact with the small cells C1 captured in the first lower capture section 141'. In this way, a horizontal cell pair (i.e., t, parallel to the bottom 100' of the microfluidic channel) is formed by the small cells C1 and the larger cells C2.

[0005] However, determining the behavior of each cell type still requires treating the cells with specific drugs.

[0006] In the microfluidic device shown in Figure 1, this selective treatment cannot be achieved because both cells are exposed to the drug flowing through the microfluidic channel. [Overview of the project]

[0007] Therefore, an object of the present invention is to provide a microfluidic device that enables the construction of a wider range of cell aggregates and the selective treatment of individual cells within the aggregate with each drug.

[0008] The present invention relates to a microfluidic device for forming a cell aggregate comprising at least one first cell and one second cell, and for individually processing selected cells of the cell aggregate: - Microfluidic channels; - At least one main inlet for the fluid containing a first cell and a second cell, respectively, located in the first portion of the microfluidic channel; - An outlet located in the second part of the microfluidic channel for controlling the flow rate of fluid within the microfluidic channel; - A first auxiliary inlet for at least one first auxiliary fluid, located in a first portion of the microfluidic channel upstream or downstream of the main inlet; - At least one cell trapping section positioned between the first and second parts within the microfluidic channel; - At least one first valve to control the flow rate of the first auxiliary fluid, causing the fluid containing the first cells and the second cells to flow at a predetermined height within the microfluidic channel, thereby guiding the first cells and the second cells to the first and second capture units, respectively. Equipped with, The objective is to provide a microfluidic device in which each cell capture section comprises at least one first capture section and one second capture section, each first and second capture section being sized to accommodate a first or second cell, the first and second capture sections being adjacent to each other in a direction perpendicular to the bottom of the microfluidic channel, forming a cell aggregate containing the captured first and second cells, and each cell being at a different height relative to the bottom of the microfluidic channel.

[0009] According to a preferred embodiment, the microfluidic device further comprises a second auxiliary inlet for at least one second auxiliary fluid located in a first portion of the microfluidic channel, and at least one second valve for controlling the flow rate of the second auxiliary fluid, the second auxiliary inlet being positioned relative to the main inlet and the first auxiliary inlet such that a fluid containing the first or second cells is sandwiched between the first and second auxiliary fluids.

[0010] Preferably, the main inlet, the first auxiliary inlet, and, if appropriate, the second auxiliary inlet are positioned at different heights relative to the bottom of the microfluidic channel, with the heights increasing along the flow direction.

[0011] It is advantageous that the main inlet, the first auxiliary inlet, and, where appropriate, the second auxiliary inlet, have a width substantially equal to the width of the microfluidic channel.

[0012] In some embodiments, the microfluidic device further comprises a filter positioned within a microfluidic channel between the first portion and at least one cell-trapping portion.

[0013] In a preferred embodiment, the microfluidic device comprises a plurality of cell-trapping sections arranged alternately within a microfluidic channel.

[0014] In some embodiments, the first and second cell capture sections of each cell capture section have different sizes, particularly different heights.

[0015] In some embodiments, at least one capture unit is configured to capture at least two cells of the same size.

[0016] Advantageously, at least one of the main inlet, the first auxiliary inlet, and optionally the second auxiliary inlet is adapted to introduce each drug into the microfluidic channel, and at least one of the first valve, and optionally the second valve, is configured such that the drug flows at a predetermined height within the microfluidic channel to selectively direct the drug to the captured first cell and / or second cell.

[0017] In some embodiments, the device further comprises an array of electrodes that are electrically insulated from each other and disposed at the bottom of the microfluidic channel, an overlapping region of two electrodes being located under each capture portion, at least one surface of each electrode being exposed within a recess of the overlapping region, and each electrode being connected to a power source to selectively apply a potential difference to the solution at each overlapping region.

[0018] In some embodiments, the device further comprises at least one pair of electrodes, each pair of electrodes being electrically insulated from each other, disposed parallel to each other at the bottom of the microfluidic channel, a region of the pair of electrodes being located under each capture portion, at least one surface of each electrode of the pair of electrodes being exposed within a recess formed around the region of the pair of electrodes, and each electrode being connected to a power source to selectively apply a potential difference to the solution at each region of the pair of electrodes.

[0019] Another object of the present invention is a method for forming a cell aggregate using the microfluidic device described above.

[0020] In the microfluidic device, the first capture portion of each cell capture portion is sized to hold the first cell, the second capture portion is sized to hold the second cell, and each cell is at a different first height and second height with respect to the bottom of the microfluidic channel.

[0021] This method comprises: - flowing a first solution containing the first cells into the microfluidic channel; -A step of using the flow of at least one auxiliary fluid to flow the first solution at a first height in the microfluidic channel to guide the first cell to the first capture unit, - A step of flowing a second solution containing a second cell into a microfluidic channel. - A step of changing the flow rate of the auxiliary fluid to control the flow of the second solution to a second height, thereby guiding the second cells to the second capture section. Includes.

[0022] In some embodiments, this method allows for the formation of cell aggregates containing at least three cells in first, second, and third capture portions located at different first, second, and third heights relative to the bottom of a microfluidic channel, using a microfluidic device comprising a main inlet and first and second auxiliary inlets. Thus, this method: - A step of flowing a first solution containing the first cells into a microfluidic channel. - A step of closing the second auxiliary inlet and using the flow of the first auxiliary fluid to flow the first solution at a first height in the microfluidic channel to guide the first cells to the first capture section. - A step of flowing a second solution containing a second cell into a microfluidic channel. -A step of using the flow of a first auxiliary fluid and the flow of a second auxiliary fluid to flow a second solution at a second height in the microfluidic channel to guide the second cells to the second capture section. - A step of flowing a third solution containing a third cell, - A step of closing the first auxiliary inlet and using the flow of the second auxiliary fluid to flow the third solution at a third height within the microfluidic channel, thereby guiding the third cells to the third capture section. Includes.

[0023] In some embodiments, the method includes the step of releasing cell aggregates from selected capture units and generating bubbles suitable for pushing cell aggregates out of the capture units by applying a potential difference to electrodes overlapping below the capture unit that is greater than the potential difference that causes electrolysis, dielectrophoresis, or electroosmosis of the solution in each recess.

[0024] In some embodiments, this method includes the step of releasing cell aggregates from a selected capture unit and generating bubbles suitable for pushing cell aggregates out of the capture unit by applying a potential difference to the electrodes of a pair of electrodes below the capture unit that is greater than the potential difference that causes electrolysis, dielectrophoresis, or electroosmosis of the solution in each recess.

[0025] Another object of the present invention is a method for selectively processing cells within a cell aggregate using the microfluidic device described above. This method is: - A step of forming a cell aggregate in the manner described above, wherein the cell aggregate includes at least one first cell located at a first height relative to the bottom of a microfluidic channel and at least one second cell located at a second height different from the first height. - A step of introducing a drug into a microfluidic channel via at least one of the main inlet, first inlet, or, where appropriate, a second inlet. - The process of introducing at least one auxiliary fluid into a microfluidic channel and directing the drug to selected cells within a cell aggregate by flowing the drug at a controlled height within the microfluidic channel through another inlet, either the main inlet, the first auxiliary inlet, or, if appropriate, the second auxiliary inlet. Includes.

[0026] In some embodiments, this method allows for the selective treatment of first and second cells of a cell aggregate with first and second drugs, respectively, using a microfluidic device comprising a main inlet and first and second auxiliary inlets. This method: - The process of flowing the buffer into the main inlet. - A step of closing the second auxiliary inlet and allowing the first drug to flow through the first auxiliary inlet so that the first drug flows at a first height within the microfluidic channel, thereby guiding the first drug to the first cell. - The process of closing the first auxiliary inlet and allowing the second drug to flow through the second auxiliary inlet so that the second drug flows at a second height within the microfluidic channel, thereby guiding the second drug to the second cell. Includes.

[0027] In another embodiment, a method for selectively treating the first and second cells of a cell aggregate with the first and second agents, respectively, is: - The process of flowing the buffer into the main inlet. - A process of flowing the first drug into the first auxiliary inlet, flowing the second drug into the second auxiliary inlet, and simultaneously flowing the first drug and the second drug in the microfluidic channel at a first height and a second height within the microfluidic channel, respectively, to guide them to the first cells and the second cells. It may include.

[0028] Advantageously, at least one of the first and second agents has a predetermined pH and / or viscosity, the pH or viscosity being selected to simulate cell interactions under predetermined conditions.

[0029] The application areas of this invention include the development of precision medicine for the treatment of dormant cancer, the evaluation of candidate molecules that stimulate immune responses, and more generally, the study of parallel cell-cell interactions for new drug discovery and / or precision drug development. [Brief explanation of the drawing]

[0030] Further features and advantages of the present invention are described in the following detailed description with reference to the accompanying drawings. [Figure 1] Figure 1 schematically illustrates the capture of two cells of different sizes by a microfluidic device described in the prior art reference [1]. [Figure 2A] Figure 2A is an overall diagram of the microfluidic device. [Figure 2B] Figure 2B is a top view of one embodiment of a microfluidic device. [Figure 2C] Figure 2C is a cross-sectional view of an embodiment of a microfluidic device. [Figure 3] Figure 3 shows the results of FEM simulations of microfluidic systems with inlets of the same height or inlets whose height increases along the flow direction. [Figure 4] Figure 4 shows cross-sectional views of the main fluid, first auxiliary fluid, and second auxiliary fluid with different flow rate conditions at the first and second inlets and outlets. [Figure 5] Figure 5 shows a series of steps for forming a cell triplet using one embodiment of a microfluidic device. [Figure 6] Figure 6 shows various capture site designs that can form cell pairs or triplets. [Figure 7] Figure 7 schematically illustrates sequential access to two cells in a cell pair (top, center) and simultaneous access to both cells (bottom) by one embodiment of a microfluidic device. [Figure 8] Figure 8 shows a side view (left) and a cross-sectional view (right) of the flow of labeled microspheres injected through the main channel within a microfluidic channel, captured by a confocal microscope, under flow conditions for continuous access to individual cell types. [Figure 9] Figure 9 shows a side view (left) and a cross-sectional view (right) obtained by confocal microscopy of the flow of labeled microspheres injected through the main channel within a microfluidic channel under flow conditions for simultaneous access to individual cell types. [Figure 10] Figure 10 shows separate access to cell types, either sequentially or concurrently. [Figure 11] Figure 11 shows specific connections between the upper cells of the aggregate, but does not show specific connections between the lower cells of the aggregate. [Figure 12] Figure 12 schematically shows an embodiment of a device equipped with an electrode array configured to generate bubbles for selectively collecting cell aggregates from the capture unit. [Figure 13] Figures 13A to 13C show embodiments of the electrode array. [Figure 14] Figure 14 schematically shows an embodiment of a device equipped with a pair of parallel electrodes configured to generate bubbles for selectively collecting cell aggregates from the capture section. [Modes for carrying out the invention]

[0031] The microfluidic device is configured to capture at least one first cell and at least one second cell to form a cell aggregate, and then to individually access selected cells of the cell aggregate.

[0032] Microfluidic devices A microfluidic device comprises a microfluidic channel configured to allow at least one fluid to flow from a first end of the channel to a second end opposite the first end. The microfluidic channel comprises a bottom extending along the longitudinal direction of the channel, which is the direction of fluid flow, two parallel side walls extending longitudinally perpendicular to the bottom, and a top surface parallel to the bottom. The distance between the side walls defines the width of the microfluidic channel. The distance between the bottom and the top surface defines the height of the microfluidic channel. The distance between the first end and the second end defines the length of the microfluidic channel. In this specification, it is assumed that the bottom is horizontal and the side walls are vertical.

[0033] The microfluidic device comprises at least one cell-capturing section positioned between the first and second ends of a microfluidic channel and configured to capture cells flowing through the fluid and form a cell aggregate. Preferably, the microfluidic device comprises a plurality of cell-capturing sections positioned in the capture region of the microfluidic channel.

[0034] In this specification, “cell aggregate” means an aggregate of at least two cells that are in contact or sufficiently close together to enable physical and / or chemical interactions between the cells. A cell aggregate consisting of two cells is called a “cell pair,” and a cell aggregate consisting of three cells is called a “cell triplet.” However, the number of cells is not limited, and a cell aggregate may consist of three or more cells. In some embodiments, the cells may be of different types. In other embodiments, at least two cells in a cell aggregate may be of the same type.

[0035] "Individually treating cells" means, as used herein, selectively delivering a drug to selected cells (or groups of cells) within a cell aggregate, while not delivering the drug to at least one other cell in the cell aggregate.

[0036] In this specification, examples of drugs include antibodies, chemotherapeutic agents, specific cell signaling modulators, growth factors, ion channel modulators, multiple coated beads, stains, dyes, nanodots, nanoparticles, and cells such as CAR-T cells and CAR-NK cells.

[0037] To enable this individual access, captured cells are stacked vertically within the capture area, that is, perpendicular to the bottom of the microfluidic channel.

[0038] Positioning cells at a specific height within the capture area and processing specific cells within a cell aggregate can be achieved through precise flow control within the microfluidic channel.

[0039] For such flow control, the microfluidic device comprises at least one main inlet for the main fluid (e.g., fluids containing first cells and second cells, respectively) and at least one first auxiliary inlet for the first auxiliary fluid. The first auxiliary inlet is positioned upstream or downstream of the main inlet along the direction of fluid flow.

[0040] Preferably, the microfluidic device further comprises at least one second auxiliary inlet for a second auxiliary fluid. The second auxiliary inlet is located at the main inlet opposite to the first auxiliary inlet. Alternatively, the main inlet is located along the longitudinal direction of the channel, between the first and second auxiliary inlets. The second auxiliary fluid may be the same fluid as the first auxiliary fluid, or it may be a different fluid.

[0041] The main inlet, the first auxiliary inlet, and the second auxiliary inlet, if present, are located at the first end of the microfluidic channel.

[0042] Each inlet may be fluidically connected to a fluid vessel or have an opening accessible to the user, allowing fluid to be directly injected into the microfluidic channel. Since different fluids may be used for cell aggregate formation and processing of individual cells, such fluidic connections are disconnectable, and each inlet can be interchangeably connected to different fluid vessels depending on the stage of use of the microfluidic device.

[0043] The microfluidic device further includes an outlet located at the second end of the microfluidic channel for controlling the flow rate of fluid within the microfluidic channel.

[0044] For this purpose, a pump may be provided at the outlet to discharge fluid from the microfluidic channel at a controlled rate.

[0045] The first and second auxiliary inlets are equipped with valves that have flow sensors, allowing auxiliary fluid to be injected into the microfluidic channels at a controlled flow rate. In some processes, the first or second auxiliary inlet may be closed.

[0046] The valve may be operated manually by an operator, or it may be operated automatically by a processor running a program.

[0047] In order to form a laminar flow of fluid within the microfluidic channel, it is preferable that the inlet has a width equal to the width of the microfluidic channel.

[0048] As will be explained in more detail below, the inlet may be positioned at a different height relative to the bottom of the microfluidic channel.

[0049] The microfluidic device may, advantageously, include a filter positioned within the microfluidic channel between the inlet and the capture region. This filter can prevent clogging of the cell capture section by aggregates or debris.

[0050] Figure 2A is a schematic diagram of such a microfluidic device 1.

[0051] From the first end 101 to the second end 102 of the microfluidic channel, the device includes a first auxiliary inlet 131, a main inlet 11, a second auxiliary inlet 132, a filter 105, a cell capture region 104, and an outlet 12. The arrows indicate the direction of flow.

[0052] Figure 2B is a top view of an embodiment of a microfluidic device.

[0053] The filter 105 may consist of a plurality of vertical columns 15 extending from the bottom to the top of the microfluidic channel and arranged alternately. The size of the columns 15 and the distance between adjacent columns along the width w of the microfluidic channel may vary along the longitudinal direction of the microfluidic channel. For example, on the inlet side, one or more rows of columns may be relatively large in size (e.g., 20 μm to 50 μm) and spaced relatively far apart (e.g., 50 μm) to block large aggregates or debris, while on the outlet side, one or more rows of columns may be relatively small in size (e.g., 10 μm) and spaced relatively far apart (e.g., 20 μm) to block small aggregates and debris.

[0054] The shape of the column is designed so as not to obstruct the fluid flow within the microfluidic channel. Specifically, the column can have a rectangular cross-section.

[0055] The capture region 104 can be configured by arranging multiple cell capture units 14 alternately to optimize cell capture. Each cell capture unit 14 is configured by arranging at least two capture units 141 and 142 vertically.

[0056] The shape of the capture area is adjusted according to the size and aggregation state (single cell or multiple cells) of the target cells.

[0057] Each capture section has two vertical side walls 144, 145 with a narrow opening 146 between them (see Figure 5), the opening being narrower than the size of the cell to prevent the captured cell from flowing through the opening.

[0058] Advantageously, the capture opening forms an angle with respect to the flow direction, for example, 45° for single-cell pairs and 30° for multiple-cell pairs. Such an angled capture opening maintains a high flow rate within the channel without disturbing the captured cells, thereby improving throughput.

[0059] The width (i.e., the internal distance between vertical side walls) and height of the capture chamber are selected based on the size of the cell being captured. Advantageously, the width of each capture chamber is slightly larger than the size of the cell being captured, thereby allowing the cell to enter the chamber without being confined. The height of each capture chamber is selected so that all cells are accommodated within the chamber while allowing for intercellular interactions.

[0060] Because the size of the aggregated cells may vary, the width and height of the capture area may change vertically. As a result, the side walls of the capture area may not be flat vertically, but may have steps separating adjacent capture areas.

[0061] Flow control Figure 2C shows a cross-sectional view of a preferred embodiment of the microfluidic device.

[0062] In this embodiment, the main inlet 11 and the first and second auxiliary inlets 131 and 132 are at different heights h from the bottom 100 of the microfluidic channel. 11 h 131 h 132 It will be placed there.

[0063] In particular, the first auxiliary inlet 131, located upstream of the main inlet 11, is positioned close to the bottom 100 of the microfluidic channel, while the second auxiliary inlet 132, located downstream of the main inlet, is positioned far from the bottom 100 of the microfluidic channel.

[0064] In other words, the first auxiliary inlet, the main inlet, and the second auxiliary inlet are designed so that their height relative to the bottom of the microfluidic channel increases in sequence.

[0065] Compared to a design in which the first auxiliary inlet, main inlet, and second auxiliary inlet are all positioned at the same height relative to the bottom of the microfluidic channel, the arrangement in Figure 2C has the advantage of achieving a uniform flow profile in a plane perpendicular to the direction of flow.

[0066] This technical effect can be easily confirmed from the comparative example in Figure 3, which shows the FEM simulation results of a microfluidic system where the inlet height is the same or the height gradually increases along the flow direction. Flow rate v at the first auxiliary inlet 131 131 , flow rate v at the second auxiliary inlet 132 132 , and the flow rate v at outlet 12 12 This is the same in all configurations.

[0067] These simulations show a non-uniform flow profile at inlets of the same height (left) in a cross-sectional view of a microfluidic channel perpendicular to the flow direction. At inlets of different heights (right), the first auxiliary inlet 131 is located at 10 μm from the bottom of the microfluidic channel, the main inlet 11 at 30 μm, and the second auxiliary inlet 132 at 35 μm, resulting in a uniform cross-sectional profile.

[0068] The first and second auxiliary inlets can be used to control the flow of the main fluid, thereby creating a "virtual" channel in which the flow of the main fluid is restricted.

[0069] Main fluid, first auxiliary fluid, and second auxiliary fluid F 11 F 131 F132 As shown in FIG. 4, which is a cross-sectional view in a direction perpendicular to the flow, this control is performed based on the thickness of the main fluid flow F in the vertical direction (upward), and / or the position of the main fluid flow F in the vertical direction (downward). FIG. 4 shows various conditions of the flow rates at the first and second inlets when the flow rate at the outlet is a constant value of 2 μl / min. 11 of the main fluid flow F, and / or the position of the main fluid flow F 11 in the vertical direction (downward). FIG. 4 shows various conditions of the flow rates at the first and second inlets when the flow rate at the outlet is a constant value of 2 μl / min.

[0070] The thickness of the main flow can be adjusted based on the flow rates of the first and second auxiliary fluids with respect to the flow rate at the outlet. The greater the flow rate at the outlet is than the flow rates at the first and second auxiliary inlets, the greater the thickness of the main flow becomes.

[0071] Furthermore, the position of the main flow can be adjusted based on the flow rate of the first auxiliary fluid with respect to the flow rate of the second auxiliary fluid. The greater the flow rate at the first auxiliary inlet is than the flow rate at the second auxiliary inlet, the higher the position of the main flow with respect to the bottom of the microfluidic channel becomes.

[0072] Therefore, by adjusting the flow rates at the first inlet, the second inlet, and the outlet, both the thickness and the height of the main flow can be finely adjusted.

[0073] When the microfluidic device has only one auxiliary inlet, the thickness of the main flow can be controlled by adjusting the flow rate of the auxiliary inlet with respect to the flow rate at the outlet. However, when there is no second auxiliary fluid flow, the main flow cannot be sandwiched between the auxiliary flows, and the vertical position cannot be controlled.

[0074] The ratio of the inlet flow rate to the outlet flow rate defines the height of the stacked flows created, regardless of the physical height of the microfluidic channel.

[0075] By precisely controlling the flows of the main fluid and the first and second auxiliary fluids in this way, aggregate cells can be stacked vertically, and individual access to the cells within the aggregate becomes possible, which will be described below.

[0076] Cell aggregate formation To form a cell aggregate, each cell type is sequentially injected into the main body from the main inlet. During the injection of each cell type, the flow rate at each auxiliary inlet is controlled relative to the flow rate at the outlet, thereby adjusting the position of the main body and, if necessary, its thickness, to guide the cells to the designated capture site in the capture section.

[0077] Figure 5 schematically illustrates the formation of a cell aggregate containing three vertically stacked cells C1, C2, and C3. Each capture site comprises three vertically stacked capture sites 141, 142, and 143. In the illustrated example, the three cells and the three capture sites are the same size. However, by controlling the flow rates of the first and second auxiliary fluids, each cell can be positioned in a selected capture site.

[0078] For example, in step (i), a mainstream body containing the first type of cell C1 is injected into the microfluidic channel from the main inlet 11. The first auxiliary inlet 131 is closed (OFF state), and auxiliary fluid is injected from the second auxiliary inlet 132 (ON state). The flow rate of the second auxiliary inlet is controlled so that the mainstream body flows into the bottom of the microfluidic channel, thereby trapping the first type of cell in the bottom trapping site 141 of each trapping unit 14.

[0079] In step (ii), a mainstream body containing the second type of cells C2 is injected into the microfluidic channel from the main inlet 11. The first and second auxiliary fluids are injected from both the first auxiliary inlet 131 and the second auxiliary inlet 132. The flow rates at the first and second auxiliary inlets are controlled so that the mainstream body flows to the center of the microfluidic channel, thereby trapping the second type of cells in the central trapping area 142 of each trapping section 14.

[0080] In step (iii), a mainstream body containing the third type of cell C3 is injected into the microfluidic channel from the main inlet 11. The second auxiliary inlet 132 is closed, and an auxiliary fluid is injected from the first auxiliary inlet 131. The flow rate of the first auxiliary inlet is controlled so that the mainstream body flows into the upper part of the microfluidic channel, thereby trapping the third type of cell C3 in the upper trapping area 143 of each trapping section 14.

[0081] Of course, the number and sequence of steps are merely illustrative and do not limit the scope of the present invention. Those skilled in the art can define the number of auxiliary fluids and the flow rate of each auxiliary fluid in a given cell aggregate.

[0082] Figure 6 shows a variety of non-limiting capture topologies that allow for the vertical aggregation of (a) cells of the same size and (b) cells of different sizes as doublets (i) or triplets (ii). Monolayer or multilayer capture units 14 (SEM image, top view) formed cell aggregates as shown in the figure and were observed with a confocal microscope (cross-sectional view). The cell aggregates formed parallel within the microfluidic channels (top view).

[0083] Individual access to cells Once a cell aggregate is formed within the capture region, it becomes possible to selectively treat one or more selected cells within the aggregate with a drug.

[0084] To this end, depending on the location of the target cells within the aggregate, the drug can be injected into the microfluidic channel via the main inlet, the first auxiliary inlet, and / or the second auxiliary inlet.

[0085] Analyzing real-time interactions between captured pairs or triplets of cells may involve exposing at least one cell from a cell aggregate to a solution with specific properties, such as pH or viscosity, to simulate cell interactions under specific conditions. Thus, by flowing one or more solutions with different properties through a microfluidic channel, it is possible to selectively access specific cells or access the entire captured cell aggregate collectively.

[0086] In some embodiments, selected cells or entire captured cell aggregates may be exposed to a pH-variable solution, particularly an acidic pH lower than that of normal tissue (approximately 7.4) (e.g., approximately 6.2–6.5). This allows for the consideration of variations present in vivo or in non-healthy environments (cancer tissue or the acidic microenvironment within bone marrow).

[0087] In other embodiments, selected cells or entire captured cell aggregates can be exposed to solutions of varying viscosities to mimic cell interactions in, for example, blood (where the viscosity of the solution is 3–5 cP (3–5 mPa·s) corresponding to the average blood viscosity) and / or bone marrow (where the viscosity of the solution is 30–38 mPa·s corresponding to the average bone marrow viscosity).

[0088] Figure 7 shows two alternative methods for selectively accessing the two cells C1 and C2 of a cell pair.

[0089] In step (i), to access the cells C1 at the bottom, the first drug A1 is injected through the first auxiliary inlet 131 and buffer B is injected through the main inlet 11. The second auxiliary inlet 132 is closed. By controlling the flow rate of the first auxiliary inlet 131 relative to the flow rate of the outlet 12, the thickness of the first drug A1 is adjusted to be less than the thickness of the cells C1 at the bottom. In this way, only the cells C1 at the bottom are treated with the first drug A1.

[0090] In optional step (ii), the upper cells C2 may be selectively treated with a second drug A2, which may be different from or identical to the first drug. To do this, the first auxiliary inlet 131 is closed, buffer B is injected from the main inlet, and the second drug A2 is injected from the second auxiliary inlet 132. The flow rate of the second auxiliary inlet 132 is controlled relative to the flow rate of the outlet 12 so that the thickness of the flow of the second drug A2 is less than the thickness of the upper cells C2. In this way, only the upper cells C2 are treated with the second drug A2.

[0091] As an alternative to the sequentially performed steps (i) and (ii), it is possible to simultaneously treat bottom cells C1 and top cells C2 with first and second drugs A1 and A2, respectively (iii). To do this, the first and second drugs A1 and A2 are injected simultaneously from the first and second auxiliary inlets 131 and 132, respectively, and buffer B is injected from the main inlet 11 to separate the first and second drugs. By controlling the flow rates of the first and second auxiliary inlets 131 and 132 relative to the flow rate of outlet 12, the thickness of the flow of the first and second drugs A1 and A2 is adjusted to be less than the thickness of the bottom cells C1 and top cells C2. In this way, each cell is treated only with the corresponding drug and not with any other drug.

[0092] Figure 8 shows side view (left) and cross-sectional view (right) of the flow of labeled microspheres injected from the main channel within a microfluidic channel under flow conditions for individual access to each cell type, as captured by a confocal microscope. The height of the "virtual" channel at the (a) bottom or (b) top of the actual microfluidic channel was varied by changing the flow rates at the first and second auxiliary inlets. Since the outlet flow was discharged at a constant flow rate (2 μl / min), the flow conditions in the capture region remained constant throughout the experiment. In (a), a higher flow rate at the first auxiliary inlet increased the height of the "virtual" channel at the bottom (flow rate at the second auxiliary inlet was 0 μl / min), and in (b), a higher flow rate at PI2 increased the height of the "virtual" channel at the top (flow rate at the first auxiliary inlet was 0 μl / min). By using the auxiliary inlets continuously, continuous access to each cell type is possible.

[0093] Figure 9 shows side views (left) and cross-sectional views (right) of the flow of labeled microspheres injected from the main channel into the microfluidic channel under flow conditions for individual access to each cell type, as captured by a confocal microscope. Compared to the experiment in Figure 8, the first inlet 131 and the auxiliary inlet 132 were used simultaneously.

[0094] Figure 10 shows sequential, individual access to a U937 cell type, for example, delivering Hoechst to the lower cells (left) and then to the upper cells (center) to stain the nuclei of these cells, or simultaneous, individual access to a U937 cell type, for example, delivering DiO to the upper cells and DiI to the lower cells (right) simultaneously to stain the membranes of both cells.

[0095] As shown in Figure 11, the nuclei of both U937 cells were stained with Hoechst stain (left). Specific binding was confirmed by selective delivery of the CD45-FITC antibody to the upper cell membrane (center). As a control, no significant binding was observed when the FITC-labeled nonspecific isotype antibody was delivered to the lower cells (circle enclosed by the dotted line) (right).

[0096] In some embodiments, after capturing and processing cell aggregates, it is possible to release selected cell aggregates by generating bubbles through electrolysis of the solution surrounding the capture area. For this purpose, the microfluidic device comprises an array of electrically insulated electrodes arranged at the bottom of the microfluidic channel. The nodes of the array (i.e., regions where two electrodes overlap) are located at the bottom of the capture area. Thus, when a sufficient potential difference (i.e., a potential difference greater than the potential difference that causes electrolysis of the solution) is applied between the two overlapping electrodes, bubbles are generated at the bottom of the microfluidic channel and rise almost vertically through the solution, pushing the cell aggregates out of the capture area. The released cell aggregates can then be moved along the microfluidic channel by the flow of the solution.

[0097] Figure 12 schematically shows an electrode array positioned at the bottom 100 of a microfluidic channel. The first electrode group E11, E12, and E13 extend perpendicular to the flow direction F, while the second electrode group E21, E22, and E23 extend parallel to the flow direction F and are therefore perpendicular to the first electrode group E11, E12, and E13. Each electrode of the first electrode group intersects with an electrode of the second electrode group below its respective trapping area. Here, "below" means that the intersection or overlapping area is in a straight line with the trapping area along a line perpendicular to the bottom of the microfluidic channel.

[0098] The first electrode group and the second electrode group are electrically insulated from each other.

[0099] Each electrode is connected to a power supply configured to selectively apply a predetermined potential. As a result, a potential difference can be generated in each crossover region.

[0100] Depending on the potential applied to each electrode, the potential difference may be greater than the potential threshold that enables electrolysis of the solution flowing through the microfluidic channel. In this case, bubbles are generated in the crossover region and rise through the solution, pushing the cell aggregates out of their respective capture points. Therefore, the cell aggregates can be recovered from the capture points without reversing the flow of the solution in the microfluidic channel or releasing the cell aggregates from other capture points.

[0101] Conversely, if the potential difference is smaller than the potential threshold, electrolysis does not occur, and the cell aggregate remains within the capture area.

[0102] For example, in the embodiment shown in Figure 12, a potential of 3V is applied to electrodes E11, E13, E21, and 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 in the crossover regions between electrodes E13 and E21 and between electrodes E13 and E23; a potential difference ΔP2=2V is applied in the crossover regions between electrodes E12 and E21 and between electrodes E11 and E22; and a potential difference ΔP3=4V is applied in the crossover region between electrodes E12 and E22. Of course, the number of each electrode group and their relative arrangement are shown for illustrative purposes only and are not intended to be limiting.

[0103] Assuming that a potential difference of at least 3V is required to induce electrolysis of the solution, the potential differences ΔP1 and ΔP2 are too low to cause electrolysis in the corresponding crossover region; however, since ΔP3 is greater than 3V, electrolysis occurs in the crossover region between electrodes E12 and E22. As a result, bubble B forms in the crossover region and rises almost vertically, pushing the cell pair C1 and C2 out of the trapping area.

[0104] The applied potential difference depends on the electrode material, electrode shape, and the ionic strength of the solution (buffer or culture medium), and can be up to 20 VD C. Alternatively, an AC signal (up to 1 or 2 MHz) can be applied to dielectrophoresis or AC electroosmosis to release cells, cell pairs, or cell triplets.

[0105] The electrode array can be fabricated by patterning a conductive material (e.g., indium tin oxide (ITO) or a metal such as gold) to form a first electrode group (e.g., a parallel electrode set) at the bottom of a microfluidic channel. The electrode thickness can reach up to 500 nm. These electrodes are covered with a first dielectric layer, the dielectric layer being thicker than the electrode thickness, thereby electrically insulating each electrode. As the dielectric material, for example, SiO2, spin-on glass, or CYTOP®, a fluoropolymer, can be used.

[0106] The second electrode group is patterned perpendicular to the first electrode group and then covered with a second dielectric layer thicker than the electrode group to electrically isolate each electrode. The width of each electrode is approximately the same as the size of the cells to be captured, for example, a few micrometers.

[0107] Next, the dielectric layer is etched around the overlapping electrode regions to form recesses in which at least a portion of each electrode is exposed to the solution flowing within the microfluidic device. Figures 13A to 13C schematically show various embodiments of such recesses.

[0108] Figure 13A shows a portion of the bottom of a microfluidic device according to one embodiment, comprising a first electrode E11 and a portion of a second electrode E21 that partially overlaps the first electrode. As described above, electrodes E11 and E21 are embedded in a first dielectric layer and a second dielectric layer (shown here as a single dielectric layer 31). The recess 300 is formed through the dielectric layer 31 to the bottom 100 of the microfluidic channel. As best seen in Figure 13B, a partial cross-sectional view of the recess in Figure 13A, etching partially exposes the top and side surfaces of the second electrode E21, as well as the top and side surfaces of the first electrode E11 on both sides of the overlapping second electrode E21. However, etching does not remove the dielectric material located between the first and second electrodes.

[0109] As shown in Figures 13A to 13B, the recess may be circular, but as shown in Figure 13C, it can be any suitable shape, such as a rectangle or a square. In the illustrated embodiment, the recess is located in the center of the overlapping region, but the position of the recess may differ as long as at least a portion of the top surface and / or side surface of each electrode is exposed.

[0110] Figure 13C shows a portion of the bottom of a microfluidic device according to another embodiment, comprising a portion of the first electrode E11 and a portion of the second electrode E21 that partially overlaps the first electrode. Similar to Figure 13A, electrodes E11 and E21 are embedded in first and second dielectric layers (shown here as a single dielectric layer 31). The recess 300 is formed through the dielectric layer 31 to the bottom 100 of the microfluidic channel. Unlike the embodiment in Figure 13A, etching exposes only the sides of the first electrode E11 and the second electrode E21. Thus, in this embodiment, the recess is formed along one side of electrodes E11 and E21. As described above, etching does not remove the dielectric material located between the first and second electrodes.

[0111] The recesses ensure that the solution comes into contact with the electrode only in the overlapping region or its vicinity. Each recess is formed directly beneath the capture area, enabling the formation of cell aggregates.

[0112] The size of the recess (e.g., diameter for a circular recess, length / width for a square or rectangular recess) is selected to generate bubbles of a suitable size for extruding cells or cell aggregates. Therefore, the size of the recess (and the resulting bubble size) is equal to or slightly larger than the cell size, which is approximately 10 μm.

[0113] In the embodiment shown in Figure 14, the electrodes are grouped in pairs, and the electrodes of each pair are arranged parallel to each other. Advantageously, as shown in Figure 14, each electrode has a linear shape parallel to the electrodes of the same pair. Advantageously, the electrodes of one pair are arranged parallel to the electrodes of another pair. The region of each electrode pair is located below each capture area. In other words, at least a portion of each electrode in the electrode pair is located below each capture area. Multiple capture areas can be arranged along each electrode pair. Advantageously, at least one face of each electrode in the electrode pair is exposed within a recess formed around the region of the electrode pair. Each electrode is connected to a power source so that a potential difference can be selectively applied to the solution in each region of the electrode pair. [Examples]

[0114] The inventors verified the efficiency of the microfluidic device in terms of cell pairing and selective processing of cell aggregates through various experiments.

[0115] manufacturing process This device consists of patterned PDMS pieces bonded to a glass coverslip. The PDMS pieces were prepared in two steps: mold fabrication using SU8 photolithography, and PDMS molding on the fabricated structure. The device is designed to have three parts: (i) an inlet area (three inlets and one filter area), (ii) a capture area, and (iii) an outlet. The inlet area consists of a central inlet for cell injection and two auxiliary inlets (first auxiliary inlet, second auxiliary inlet) for reagent supply. The filter area has vertical columns with a diameter of 50 μm, forming an array with 50 μm gaps between rows and columns, allowing single cells to pass through safely while preventing large aggregates and fragments from reaching the mechanical capture area. The capture area is where cells aggregate. The shape of the capture area varies depending on the size of the target cells and the state of aggregation (single or multiple cells). As the final part, the outlet is connected to a pressure pump to provide a constant flow to the capture area so that cell activity can be monitored under stable conditions.

[0116] The molded body was fabricated by patterning four layers of SU8 on a silicon wafer to have channels with a height of 50 μm for triplet demonstration, channels with a height of 38 μm for doublet demonstration, and channels with a height of 30 μm for demonstration of aggregates of different cell sizes. The first layer corresponds to a continuous flow layer, excluding the filter columns and support columns for the capture array. The second and fourth layers of SU8, corresponding to the first and second auxiliary inlets, have fixed thicknesses, i.e., 10 μm and 5 μm, respectively. The third layer, used as the main inlet, has a thickness that depends on the total height of the channels. The first layer, 2 μm thick, was fabricated by spin-coating SU8 2002 on a 3-inch silicon wafer (3500 rpm, 30 seconds). After soft baking (95°C for 2 minutes), the wafer was exposed (375 nm laser, Heidelberg MLA-150, maskless lithography system at 100 mJ / cm²) 2 The wafer was irradiated (at a certain dose) and post-baked (at 95°C for 2 minutes). The second layer forming the first auxiliary entrance was fabricated with SU8 3010. The resist was spin-coated (4000 rpm, 30 seconds) to a thickness of 8 μm. The wafer was then soft-baked (at 65°C for 1 minute, at 95°C for 10 minutes) and exposed (150 mJ / cm²). 2 The wafers were soft baked (65°C for 2 minutes, 95°C for 5 minutes) and post-baked. The main inlet and the third layer forming the first layer of the multilayer trapping array were fabricated with SU8 3025. The resist was spin-coated at 2000 rpm and 4300 rpm for 30 seconds so that the thickness of the single-layer trapping arrays (cell triplet and doublet, respectively) was 35 μm and 23 μm, respectively. For multilayer trapping arrays, SU8 3005 was applied to a thickness of 15 μm at 800 rpm. The wafers were soft baked (65°C for 1 minute, 95°C for 15 minutes) and exposed (220 mJ / cm²). 2 The wafer was then post-baked (2 minutes at 65°C, 5 minutes at 95°C). The fourth layer forming PI2, the filter region, and the capture region (the second layer of the multilayer capture region array) was spin-coated with SU8 2005 to a thickness of 5-8 μm (1000-2500 rpm, 30 seconds). The coated wafer was soft-baked (1 minute at 65°C, 5 minutes at 95°C) and exposed to 220 mJ / cm².2 The PDMS layers were then coated and post-baked (1 minute at 65°C, 5 minutes at 95°C). After coating all layers, the photoresist was developed with SU8 developer and hard-baked (15 minutes at 150°C). Finally, a thin Teflon film was deposited (Oxford PlasmaPro80, 100W, C4F8, 30m Torr, 30 seconds) to allow for easy removal of the PDMS fragments after molding.

[0117] A PDMS mixture (base elastomer to curing agent in a 10:1 ratio) was degassed and poured onto a silicon wafer having an SU8 structure. The thickness of the PDMS was approximately 1.5 mm to limit the volume of the central inlet reservoir to an appropriate value for rapid injection of the cell suspension. After curing at 70°C for 5 hours, the cured PDMS was peeled off and cut. The first and second auxiliary inlet and outlet openings were fabricated using a 0.5 mm diameter biopsy puncher (for connection to a pressure pump). The main inlet opening was fabricated using a 1.5 mm diameter biopsy puncher. The PDMS was then bonded to a glass coverslip (0.17 mm thick) by activating the bottom surface of the PDMS with a plasma cleaner (Harrick, Hi-level, 5 minutes) and firing at 90°C for 30 minutes. The top surface of the PDMS was covered with tape to protect its inherent hydrophobic surface properties, which may be altered during plasma cleaning.

[0118] Setup and Protocol The inventors used (i) a confocal microscope (ZEISS LSM880) for experiments requiring 3D visualization, and (ii) an inverted microscope (Olympus IX83) for characterizing pairing efficiency.

[0119] A microfluidic device mounted on a microscope stage was connected to a pressure pump (Fluigent, LineUp® Push-Pull) via three controllers. Two controllers were connected to the first and second auxiliary inlets, and a third controller was connected to the outlet. Each controller was equipped with a dedicated flow sensor (Fluigent, FLU-MD) to precisely control the liquid flow rate (inlet / outlet). The auxiliary inlet controllers always operated in injection mode, injecting solution into the channels. The outlet controllers, on the other hand, always operated in recovery mode, draining solution from the device.

[0120] Before the experiment, the microfluidic device was treated with Pluronic (F-127) solution (50 mg / mL) to prevent nonspecific adhesion to the channel surface. A syringe pump (Kd Scientific) was connected to the outlet, and the fluid was injected at a flow rate of 25 μl / min for 3 minutes and then at 5 μl / min for 7 minutes. Deionized water was then injected from both PIs (25 μl / min) and discharged from the outlet (20 μl / min) for 10 minutes of washing. The flow rate at the outlet was smaller than that of the auxiliary inlet, creating a backflow from the auxiliary inlet to the main inlet to wash the main inlet reservoir. Using the same washing configuration, the channels were filled with culture medium from the first and second auxiliary inlets. An alternative PDMS layer (thickness <1 mm) with a diameter 1.2 mm opening was placed on the main inlet to maintain the device's inherent hydrophobic surface properties. This PDMS piece helped prevent sample spillage from the main inlet during the experiment. After surface treatment, the device was placed on a microscope stage and the microfluidic connection was fabricated.

[0121] The stacked flows were characterized using 0.50 μm fluorescent polymer microspheres (FSDG003, Bangs Laboratories, Inc.). Different flow conditions were used to control the size of each flow (Figure 4 (top)). Similarly, by controlling the flow at the auxiliary inlet and maintaining the outlet flow at 2 μl / min, thin "virtual" channels could be positioned along the channel height (Figure 4 (bottom), Figures 8 and 9). Monitoring the stacked flow conditions of passive structures within the channel—i.e., before the filter, between the filter and the capture section, and after the capture section—showed that these structures had little effect on the stacked flow.

[0122] The first step of the experimental protocol was the aggregation of cells in the capture section. Throughout the experiment, in all capture section topologies, the outlet discharged liquid from the channel at a constant flow rate of n (2 μl / min). The inventors injected cells (150,000 cells / ml, Table 1) from the main inlet while adjusting the flow conditions of the auxiliary inlet according to the target aggregate shape (Table 2). The flow conditions were maintained for 5 minutes for each cell layer, during which a 5-minute wash was performed.

[0123] [Table 1]

[0124] [Table 2]

[0125] The second step is individual access to cells within the aggregate. Auxiliary inlet and outlet flows enable individual or simultaneous access to cells. Flow stability in the capture region was maintained by keeping the outlet flow condition (2 μl / min) constant. By varying the flow conditions of the auxiliary inlets, it was possible to deliver molecules / drugs to different parts of vertically aggregated cells.

[0126] Individual access can be performed sequentially for each cell type using only one auxiliary inlet. Hoechst (1 μg / ml) was delivered to the lower cells for 10 minutes from the first auxiliary inlet (0.5 μl / min for the first auxiliary inlet, 0 μl / min for the second auxiliary inlet). Then, the first auxiliary inlet was set to 0 μl / min, the channel was washed with culture medium for 5 minutes, and then injected from the main inlet. The inventors delivered Hoechst to the upper cells via the second auxiliary inlet (0.5 μl / min) while maintaining the first auxiliary inlet at 0 μl / min. After a 10-minute flow, the channel was washed again. On the other hand, simultaneous access required the use of both auxiliary inlets together. After nuclear staining, the inventors simultaneously injected DiI (1 μM) into the first auxiliary inlet and DiO (1 μM) into the second auxiliary inlet at a flow rate of 0.5 μl / min for 5 minutes to stain the cell membranes. DiI stained the bottom of the cells at the bottom, and DiO stained the top of the cells at the top. The inventors used a microfluidic switch (2-Switch Fluigent) at an auxiliary inlet to inject the necessary solution (culture medium for the aggregate, or drug / staining agent for accessing the cells) into the channel.

[0127] Specific access to specific cells did not necessarily have to be limited to the upper part of upper cells or the lower part of lower cells. By injecting molecules / drugs using both auxiliary and primary entry points, access to the intermediate regions of cells became possible. We tested both access types using specific binding by immunocytofluorescence staining. We demonstrated the former protocol targeting lower cells by injecting a FITC-labeled anti-CD45 antibody solution (1 μg / mL) through a first auxiliary entry point (0.2 μl / min, 5 minutes). After washing with culture medium injected through the primary entry point for 5 minutes, upper cells were exposed as a control to isotype FITC (1 μg / mL) injected through a second auxiliary entry point (0.5 μl / min, 5 minutes). The results showed that only the specific binding condition resulted in successful staining, which was confirmed by changing the order of staining, i.e., by using CD45-FITC on upper cells and isotype FITC on lower cells (Figure 11). The latter protocol, which accesses the central portion of the cell, was demonstrated by delivering CD45-FITC through the main inlet and flowing culture medium at 1.2 μl / min and 0.3 μl / min through the first and second auxiliary inlets, respectively (Figure 7. Controls with isotype FITC were performed under corresponding conditions).

[0128] In cell activity analysis, the multilayer cell doublet structure was demonstrated using continuous access to aggregated cells. Before the experiment, cells were incubated with RPMI medium diluted with Fluo4 (2 μM) at 37°C for 30 minutes. After centrifugation, the cells were washed and suspended in an extracellular solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM Hepes) and reacted with the calcium ionophore ionomycin. As described above, cells were aggregated by continuous injection of cell types. First, KG1 cell line (150,000 cells / ml) was injected for 5 minutes through the main inlet (first auxiliary inlet: 0 μl / min, second auxiliary inlet: 0.5 μl / min, outlet: 2 μl / min). Then, primary human CD8 +T lymphocytes (150,000 cells / ml) were injected for 5 minutes through the main inlet (first auxiliary inlet: 1.5 μl / min, second auxiliary inlet: 0 μl / min, outlet: 2 μl / min). Confocal images were taken every 3 minutes to monitor cell activity. Ionomycin (10 μg / mL) was delivered to the lower cells through the first auxiliary inlet for 7 minutes (0.5 μl / min), and the second auxiliary inlet was set to 0 μl / min. After washing with ionomycin for 5 minutes (t=12 min), this process was repeated for the upper cells, with the flow rate of the second auxiliary inlet changed to 0.2 μl / min and the flow rate of the first auxiliary inlet changed to 0 μl / min. Then, the cells were washed again with ionomycin for 5 minutes (t=27 min).

[0129] Finite element modeling Prior to manufacturing, the device was modeled and optimized using the finite element method (FEM). The microchannel geometry was plotted using the COMSOL Multiphysics (v.5.5) platform. Subsequently, simulations were performed using the same geometry as the actual device. The constructed model utilizes two physical properties: laminar flow and diluent transport. Both of these physical properties were incorporated into the microfluidic module and analyzed in the fluid domain. The flow behavior was obtained by solving the Navier-Stokes equations for an incompressible Newtonian fluid. In the fluid domain, an incompressible Newtonian fluid has a low Reynolds number (Re<1) and is described by a coupled system of the Navier-Stokes equations.

number

[0130] The accumulated flow was simulated by solving the transport physics of dilute materials. Material concentrations were monitored using confident diffusion equations.

number

[0131] [Table 3]

[0132] cell preparation The experiment included human leukemia cell lines (KG1, U937), PBMCs, and CD8 + Various cell types, including T lymphocytes, were used.

[0133] KG1 cell line (CCL-246™-ATCC™) was cultured in RPMI1640 medium (Gibco, Waltham, Massachusetts, USA) supplemented with 1% penicillin-streptomycin antibiotic cocktail (Gibco) and 20% fetal bovine serum (FBS) (Gibco).

[0134] All cells were stored in a 37°C incubator under a high-humidity atmosphere (~95%) supplied with CO2.

[0135] U937 cells (CCL-246® and CRL-1593.2®, ATCC®) were cultured according to the same process as used for the KG1 cell line, except that the amount of FBS was reduced to 10%.

[0136] PBMCs were purified and isolated from the blood of healthy donors using Ficoll-Hypaque density gradient centrifugation. EasySep® Human CD8 + Using a T cell isolation kit and column-free magnetic cell isolation method (Stem Cell Technology), human primary cultured CD8 cells were separated. + T lymphocytes were negatively selected from PBMCs.

[0137] CD8 + T lymphocytes were activated by culturing them for 3 days with Dynabeads® Human T-Activator CD3 / CD28 (Gibco) in a 1:1 ratio, supplemented with 0.1 μg / ml of IL-2 (Ozyme).

[0138] Three days later, the anti-CD3 / CD28 coated Dynabeads were removed using a magnetic separation column (Stem Cell Technology).

[0139] activated CD8 + T lymphocytes were resuspended in RPMI1640 medium (Gibco, Waltham, Massachusetts, USA) supplemented with a 1% penicillin-streptomycin antibiotic cocktail (Gibco) and 10% FBS (Gibco).

[0140] Furthermore, the inventors added 0.1 μg / ml of IL-2 and incubated it for 7 days.

[0141] The cell solution was treated with DNase (50 U / ml) to prevent long DNA segments from adhering to the microfluidic device.

[0142] The remaining aggregates were removed using a 30 μm pore filter (MACS® SmartStrainers), and the solution was converted into a single-cell suspension before injection into the device.

[0143] In situ staining and stimulation of single cells DiI and DiO (manufactured by Life Technologies) were prepared according to the instructions of the cell membrane staining manufacturer.

[0144] To stain the cell nuclei, a Hoechst (33342, NucBlue® Live Ready Probes®) solution (1 μg / mL) was prepared.

[0145] Antibody staining was performed by perfusion with anti-CD45 antibody conjugated to FITC (Invitrogen).

[0146] In the control experiment, the corresponding isotype bound to FITC was used (Invitrogen).

[0147] Intracellular calcium concentration was measured using the fluorescent calcium dye Fluo-4AM (Invitrogen).

[0148] In situ stimulation was performed by loading cells onto a serum-free RPMI1640 (Gibco, phenol red-free) at 37°C and 5% CO2 for 45 minutes.

[0149] Next, the cells were washed before use.

[0150] References [1]F. A. Shaik et al., “Pairing cells of different sizes in a microfluidic device for immunological synapse monitoring,” Lab Chip, vol. 22, no. 5, pp. 908-920, Mar 1 2022, doi: 10.1039 / d1lc01156a

Claims

1. A microfluidic device (1) for forming a cell aggregate comprising at least one first cell (C1) and one second cell (C2), and for individually processing selected cells of the cell aggregate: - Microfluidic channel (10); - At least one main inlet (11) for fluids containing a first cell and a second cell, respectively, located in the first portion (101) of the microfluidic channel; - An outlet (12) located in the second portion (102) of the microfluidic channel for controlling the flow rate of fluid within the microfluidic channel; - A first auxiliary inlet (131) for at least one first auxiliary fluid, located in the first portion (101) of the microfluidic channel upstream or downstream of the main inlet (11); - At least one cell-trapping portion (14) positioned between the first and second portions within the microfluidic channel; - At least one first valve for controlling the flow rate of the first auxiliary fluid to guide the fluid containing the first cells and the second cells, respectively, through the microfluidic channel at a first predetermined height and a second predetermined height, thereby guiding the first cells and the second cells to a first capture unit and a second capture unit, respectively. Equipped with, Each cell capture section (14) comprises at least one first capture section (141) and one second capture section (142), each first and second capture section being sized to accommodate a first or second cell, the first and second capture sections being adjacent to each other in a direction perpendicular to the bottom (100) of the microfluidic channel, forming a cell aggregate containing the captured first and second cells, each cell being at a different height relative to the bottom of the microfluidic channel, the microfluidic device.

2. The microfluidic device according to claim 1, further comprising a second auxiliary inlet (132) for at least one second auxiliary fluid located in a first portion (101) of the microfluidic channel, and at least one second valve for controlling the flow rate of the second auxiliary fluid, wherein the second auxiliary inlet (132) is positioned relative to the main inlet (11) and the first auxiliary inlet (131) such that a fluid containing the first or second cells is sandwiched between the first and second auxiliary fluids.

3. The main inlet (11), the first auxiliary inlet (131), and, where appropriate, the second auxiliary inlet (132) are at different heights (h) relative to the bottom (100) of the microfluidic channel. １１ , h １３１ , h １３２ The microfluidic device according to claim 1 or 2, wherein the height increases along the flow direction, and is positioned in a )

4. A microfluidic device according to any one of claims 1 to 3, wherein the widths of the main inlet (11), the first auxiliary inlet (131), and, where appropriate, the second auxiliary inlet (132) are substantially equal to the width (w) of the microfluidic channel (10).

5. The microfluidic device according to any one of claims 1 to 4, further comprising a filter (15) disposed within a microfluidic channel (10) between a first portion (101) and at least one cell-trapping portion (14).

6. The microfluidic device according to any one of claims 1 to 5, comprising a plurality of cell-trapping units (14) arranged alternately within the microfluidic channel.

7. The microfluidic device according to any one of claims 1 to 6, wherein the first and second capturing sections (141, 142) have different sizes, in particular different heights.

8. The microfluidic device according to any one of claims 1 to 6, wherein at least one capture portion (141, 142) is configured to capture at least two cells of the same size.

9. The microfluidic device according to any one of claims 1 to 8, wherein at least one of the main inlet, the first auxiliary inlet, and optionally the second auxiliary inlet is adapted to introduce each drug (B, A1, A2) into the microfluidic channel, and at least one of the first valve, and optionally the second valve is configured to control the flow rate of the drug so that the drug flows at a predetermined height within the microfluidic channel in order to selectively guide the drug to the captured first and / or second cells.

10. The microfluidic device according to any one of claims 1 to 9, further comprising an array of electrodes (E11, E12, E13, E21, E22, E23) electrically insulated from each other and positioned at the bottom (100) of a microfluidic channel, wherein overlapping regions of two electrodes are located beneath each capture portion, at least one surface of each electrode is exposed within a recess (300) formed around 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.

11. A microfluidic device according to any one of claims 1 to 10, further comprising at least one pair of electrodes (E11, E12, E13, E21, E22, E23), wherein each pair of electrodes is electrically insulated from one another, is arranged parallel to one another at the bottom (100) of a microfluidic channel, the region of each pair of electrodes is located beneath each capture portion, at least one surface of each electrode of the pair of electrodes is exposed in a recess (300) formed around the region of the pair of electrodes, and each electrode is connected to a power source to selectively apply a potential difference to a solution in the region of each pair of electrodes.

12. A method for forming a cell aggregate using a microfluidic device (1) according to any one of claims 1 to 11, wherein a first capture portion (141) is sized to hold a first cell (C1), a second capture portion (142) is sized to hold a second cell (C2), and each cell is at a different first and second height relative to the bottom (100) of the microfluidic channel: - A step of flowing a first solution (F11) containing a first cell (C1) into the microfluidic channel, - A step of using the flow of at least one auxiliary fluid (F131, F132) to flow the first solution at a first height in the microfluidic channel to guide the first cell (C1) to the first capture unit (141), - A step of flowing a second solution (F11) containing a second cell (C2) into the microfluidic channel, - A step of changing the flow rate of auxiliary fluids (F131, F132) to control the flow of the second solution to a second height, thereby guiding the second cell (C2) to the second capture unit (142). The method, including the method described above.

13. A method according to claim 12, wherein a cell aggregate containing at least three cells (C1, C2, C3) is formed in first, second, and third capture portions (141, 142, 143) located at different first, second, and third heights relative to the bottom (100) of the microfluidic channel, wherein the microfluidic device according to claim 2 is used. - A step of flowing a first solution (F11) containing a first cell (C1) into the microfluidic channel, - A step of closing the first auxiliary inlet (131) and using the flow of the second auxiliary fluid (F132) to flow the first solution (F11) at a first height in the microfluidic channel to guide the first cell (C1) to the first capture unit (141), - A step of flowing a second solution (F11) containing a second cell (C2) into the microfluidic channel, - A step of using the flow of the first auxiliary fluid (F131) and the flow of the second auxiliary fluid (F132) to flow the second solution at a second height in the microfluidic channel and guide the second cell (C2) to the second capture unit (142), - A step of flowing a third solution (F11) containing a third cell (C3), - The process of closing the second auxiliary inlet (132) and using the flow of the first auxiliary fluid (F131) to flow the third solution at a third height in the microfluidic channel, thereby guiding the third cell (C3) to the third capture unit (143). The method, including the method described above.

14. A method according to any one of claims 12 to 13, performed using the device according to claim 10, further comprising the step of releasing cell aggregates from a selected capture portion and generating bubbles (B) suitable for pushing cell aggregates out of the capture portion by applying a potential difference greater than the potential difference that causes electrolysis, dielectrophoresis, or electroosmosis of the solution in each recess (300) to electrodes (E11, E21) that overlap below the capture portion.

15. A method according to any one of claims 12 to 13, performed using the device according to claim 11, further comprising the step of releasing cell aggregates from a selected capture unit and generating bubbles (B) suitable for pushing cell aggregates out of the capture unit by applying a potential difference greater than the potential difference that causes electrolysis, dielectrophoresis, or electroosmosis of the solution in each recess (300) to the electrodes (E11, E21) of a pair of electrodes below the capture unit.

16. A method for selectively processing cells within a cell aggregate, - A step of forming a cell aggregate by the method of claim 12 or 13, wherein the cell aggregate includes at least one first cell (C1) located at a first height relative to the bottom (100) of a microfluidic channel and at least one second cell (C2) located at a second height different from the first height. - A step of introducing drugs (A1, A2) into a microfluidic channel via at least one of the main inlet, first inlet, or, where appropriate, second inlet. - A step of introducing at least one auxiliary fluid (B) into a microfluidic channel and directing the drug to selected cells in a cell aggregate by flowing the drug at a controlled height within the microfluidic channel through another inlet, either the main inlet, the first auxiliary inlet, or, if appropriate, the second auxiliary inlet. The method further includes the method described above.

17. A method according to claim 16 for selectively treating first and second cells (C1, C2) of a cell aggregate with first and second drugs (A1, A2), respectively, using the microfluidic device described in claim 2, - Process of flowing buffer (B) into the main inlet, - A step of closing the second auxiliary inlet (132) and allowing the first drug (A1) to flow through the first auxiliary inlet so that the first drug (A1) flows at a first height within the microfluidic channel, thereby guiding the first drug (A1) to the first cell (C1), - The process of closing the first auxiliary inlet (131) and allowing the second drug (A2) to flow through the second auxiliary inlet so that the second drug (A2) flows at a second height within the microfluidic channel, thereby guiding the second drug (A2) to the second cell (C2). The method, including the method described above.

18. A method according to claim 16 for selectively treating first and second cells (C1, C2) of a cell aggregate with first and second drugs (A1, A2), respectively, using the microfluidic device described in claim 2, - Process of flowing buffer (B) into the main inlet, - A process of flowing the first drug (A1) into the first auxiliary inlet, flowing the second drug (A2) into the second auxiliary inlet, and simultaneously flowing the first drug (A1) and the second drug (A2) at a first height and a second height within the microfluidic channel, respectively, to guide them to the first cell (C1) and the second cell (C2). The method, including the method described above.

19. The method according to any one of claims 16 to 18, wherein at least one of the first and second agents has a predetermined pH and / or a predetermined viscosity, the pH or viscosity being selected to simulate cell interactions under predetermined conditions.

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