Microfluidic chip

Abstract

The microfluidic device (100) comprises a substrate and functional units (102). Each functional unit (102) comprises a first chamber (104) arranged in fluid communication with the first channel (106) and a second chamber (108) arranged in fluid communication with the second channel (110). A microchannel array (114) is arranged to connect the first channel (106) and the second channel (108). A method of culturing cells includes providing immune cells to the first chamber (104), providing cancer cells to the second chamber (108), incubating the device (100), and counting the immune cells and / or the cancer cells while observing migration of the immune cells to the cancer cells. In this manufacturing method, a substrate is provided and cast to define functional units. Each functional unit comprises a first chamber arranged in fluid communication with the first channel and a second chamber arranged in fluid communication with the second channel. The microchannel array is disposed so as to connect the first channel and the second channel.

Description

[Technical Field]

[0001] The present application relates to a device for culturing cells. More specifically, the present application relates to a device for culturing cells, spheroids, and organoids in vitro. [Background Art]

[0002] Microfluidic chips have been applied as a solution for studying the efficacy of immunotherapy by evaluating immune cell migration toward cancer cells. One state-of-the-art chip was developed by Businaro et al. (Lab Chip 13 (2013) 229-239) and has been used to test the effects of several immunotherapeutic approaches (e.g., Lucarini et al. J. Investig. Dermatol. 137 (2017) 159-169). In these assays, Matrigel has often been used to provide a three-dimensional environment for cancer cells. Furthermore, fully human in vitro microfluidic chip assays have been used for testing immunotherapy aimed at personalized medicine (Al Samadi et al., Experimental Cell Research 383 (2019) 111508). In general, microfluidic chip designs have been used to test both the migration of immune cells toward cancer cells and their cytotoxic activity, regardless of the presence or absence of immunomodulators. Chip design is important: a sufficiently large sample volume is required for accurate and reproducible measurements, while the chip needs to be miniaturized to fit on a microscope slide for imaging and measurement. Current designs such as that of Businaro et al. only allow measurement of a single sample on the chip.

[0003] Therefore, conventional devices may offer only limited possibilities for imaging, and these devices can also be cumbersome to load. Traditionally, microfluidic chip devices are loaded manually by individually loading each chamber with a pipette, which is time-consuming, laborious, and / or prone to human error. Attempting to load the devices with automated pipettes typically results in problems with air bubble formation, making automated pipetting unreliable. Furthermore, known devices have limited gas exchange and restricted nutrient supply to cells. [Overview of the project]

[0004] The object of the disclosed invention is to provide an improved device and to alleviate at least some of the problems associated with prior art cell culture devices.

[0005] According to one aspect of this application, a microfluidic device comprises a gas-permeable substrate and one or more functional units. Each functional unit comprises two first chambers arranged to be fluidly connected by a first channel. Each functional unit comprises two second chambers arranged to be fluidly connected by a second channel. Each functional unit comprises a microfluidic array arranged to connect the first channel and the second channel. The microfluidic array, the portion of the first channel fluidly connected to the microfluidic array, and the portion of the second channel (110) fluidly connected to the microfluidic array form the operating portion of each functional unit (102).

[0006] According to another aspect of this application, each functional unit of the microfluidic device may be an isolated unit. This means that any one functional unit is not in fluid communication with any of the other functional units. Each functional unit may be arranged to be loaded independently. The width of the microfluidic array can be in the range of 30 μm to 9000 μm, preferably 3000 μm to 7000 μm, more preferably 4000 μm to 6000 μm. Alternatively, the width of the microfluidic array can be in the range of 30 μm to 2000 μm, preferably 50 μm to 100 μm. The microfluidic array includes microchannels, each microchannel having a width of 10 μm to 15 μm, preferably 11 μm to 13 μm, a height of 5 μm to 20 μm, preferably 10 μm to 15 μm, a length of 10 μm to 2000 μm, preferably 10 μm to 1000 μm, and a spacing of 20 μm to 40 μm between each microchannel.

[0007] According to another aspect of this application, each of the first chambers of the microfluidic device has a height of 100 μm to 3 cm and an essentially circular cross-section having a radius of 0.5 mm to 10 mm, preferably 3 mm to 5 mm, or 0.7 mm 2 ~314mm 2 Preferably 28 mm 2 ~78.5mm 2It can have any other two-dimensional shape having an area spanning over a certain distance. Each of the second chambers of the microfluidic device may have a height of 100 μm to 3 cm and a surface area corresponding to an essentially circular cross-section, with a radius of 200 μm to 400 μm, or a radius corresponding to the outer diameter of the loading device used to load each of the second chambers. The first channel of the microfluidic device may have a height of 10 μm to 300 μm, preferably 170 μm to 190 μm, for example 189 μm, and a width of 200 μm to 3000 μm, preferably 1190 μm. The second channel of the microfluidic device may have a height of 10 μm to 300 μm, preferably 170 μm to 190 μm, for example 189 μm, and a width of 100 μm to 1000 μm, preferably 490 μm. The length of the first channel of the microfluidic device can be at least the length of the microfluidic array, and the length of the second channel of the microfluidic device can be at least the length of the microfluidic array.

[0008] According to another aspect of this application, at least a portion of the microfluidic device, or optionally all or most of the microfluidic device excluding the first and second chambers, may be covered with a transparent material.

[0009] According to one aspect of this application, the microfluidic device may be configured to be placed on a slide for microscopic examination. According to another aspect of this application, the microfluidic device may be configured to be placed on a microplate. Depending on the experiment, the microfluidic device may include 1 to 384 functional units.

[0010] According to one aspect of this application, a microfluidic device may include PDMS. PDMS or a suitable derivative of PDMS can provide appropriate gas permeability, which facilitates important gas exchange in cell culture.

[0011] According to the cell culture method, the microfluidic device comprises at least one first chamber containing at least immune cells and at least one second chamber containing at least cancer cells. The microfluidic device is incubated, and then the immune cells and / or cancer cells are counted while observing the migration of immune cells to cancer cells. At least immune cells for at least one first chamber, and / or at least cancer cells for at least one second chamber, are supplied by one or more automated pipettes.

[0012] According to the method for manufacturing a microfluidic chip, a substrate for a microfluidic chip is provided. The thickness of the substrate is limited to 30 μm to 3.5 mm. The substrate is processed to define one or more functional units. Each functional unit comprises two first chambers arranged to be fluidly connected by a first channel. Each functional unit comprises two second chambers arranged to be fluidly connected by a second channel. Each functional unit comprises a microchannel array arranged to connect the first channel and the second channel. Each functional unit comprises an operating portion arranged to connect a portion of the first channel that is fluidly connected to the microchannel array and a portion of the second channel that is fluidly connected to the microchannel array (114). According to this method, the substrate is then cured.

[0013] Conventional automated pipetting solutions have long channels. Long channels cause a large pressure drop during pipetting. Shorter channels result in a smaller pressure drop. A smaller pressure drop allows for better control of the liquid flow when injecting the liquid into the device. This is an important point because automated pipettes are designed to "drop" liquids with minimal resistance, not to inject them into cavities where back pressure is present. Therefore, more viscous liquids typically used in cell culture experiments can be difficult to inject into microfluidic channels. For these reasons, shortening the channel results in a more reliable filling process and a reduced possibility of bubble formation during filling. Another point is that if the sample volume required is small, less drug is needed for the test. This is a clear advantage in reducing the cost of some in vitro tests, such as those involving immune checkpoint inhibitors or other antibody-based therapies, where drug costs can be significant.

[0014] Another improvement in the operation of the new device is the priming time of the microchannel, i.e., the faster disappearance of the air pockets initially present in the microchannel after the liquid is loaded from both sides, thus enabling faster operation of the device.

[0015] The main difference between the disclosed solution and prior art is the improved device structure (referring to the arrangement, size, shape, and / or dimensions of the flow channels and / or chambers), in particular at least the flow channel structure, which enables the arrangement of multiple (e.g., up to 384) functional units on a single chip. Each functional unit comprises two first chambers connected by a first flow channel and two second chambers connected by a second flow channel. The first and second flow channels are connected by a microfluidic array. In this specification, the term “connected” may generally mean fluid communication.

[0016] The inventors have discovered that the conventional side chambers and side channels included for filling with fresh culture medium may be unnecessary to provide sufficient cell viability. Eliminating these channels can result in eliminating or reducing the influence of nutrient concentration gradients formed during device operation from the opposite direction in which cells attempt to migrate, driven by chemical signals originating from the other side of the microfluidic array. Therefore, the device according to the present invention can be realized without such side channels and can reduce nutrient concentration gradients.

[0017] Conventional technologies assume that a large side container / side chamber is necessary to provide sufficient nutrients to cells. This has prevented the mounting of more functional units on a slide. In the microfluidic device disclosed with this configuration, due to the shape of the chamber and / or channel, specifically its height and / or volume, more liquid per unit volume of cells can be loaded into the chamber and channel, enabling sufficient nutrient supply while maintaining an overall compact size of the functional unit. The liquid can be, for example, Matrigel or other viscous media with added gel-forming additives such as fibrinogen. Conventional solutions have so far used a volume of 150 μL. In the configuration proposed herein, the total volume can be 1 μL to 1000 μL, preferably 1 μL to 500 μL, and more preferably 1 μL to 100 μL. In the conventional cell suspension, the cell content is 3 μL for cancer cells and 150 μL for immune cells. In this configuration, the cell content of the cell suspension volume can be 0.1 μL to 149 μL, preferably 0.1 μL to 2.9 μL, more preferably 0.5 μL to 2 μL, and even more preferably 1 μL to 2 μL.

[0018] The liquid can be a cell culture medium. The cell culture medium is selected based on the type of cell or organoid and the assay. Generally, all cell culture media contain a carbon source as an energy source, amino acids as building blocks for proteins, and vitamins that promote cell survival and growth. Furthermore, the equilibrium salt solution of the medium needs to maintain an isotonic mixture of ions to optimize intracellular osmotic pressure and supply essential metal ions that act as cofactors for enzymatic reactions. Additionally, a buffer (e.g., bicarbonate or HEPES) is used to maintain a balanced pH in the medium.

[0019] It is important that the liquid to be loaded is designed to form a hydrogel suitable for three-dimensional cell culture. These hydrogels may be selected from a list including, but are not limited to, nanocrystals (i.e., nanofibrillated cellulose), chitosan, alginates, hyaluronans, polypeptides, collagen, gelatin, fibrinogen, agarose, or any combination thereof or functionalized versions based on these complex forms. Preferred synthetic hydrogels may be, for example, polyethylene glycol, polyurethane, poly(ε-caprolactone), or any other functionalized versions thereof or combinations thereof. Other components of the culture medium may include, for example, laminin, entactin, heparan sulfate proteoglycans, tumor growth factors, etc.

[0020] To evaluate the efficacy of the drug, drugs such as pembrolizumab, nivolumab, cemiprimab, atezolizumab, avelumab, durvalumab, and / or any other monoclonal antibody-based drugs having the ability to block receptors on cancer cells or immune cells for direct or indirect therapeutic purposes may be added to the cell suspension.

[0021] It is important that hydrogel formation occurs after the cell suspension is injected into the microfluidic channel. Gel formation can be initiated by heat or light. After injecting a cell suspension at a low temperature (4-25°C) into the chamber, the device may be placed at 37°C. Heat initiates gel formation, creating a hydrogel that mimics the cancer microenvironment.

[0022] Each functional unit may be isolated, that is, it may not be in physical contact with any other functional unit of the device. Therefore, each functional unit may be suitable for independently loading any type of sample. This is an advantage over the prior art in which the two sides can communicate through a central channel, but the use of cell suspensions loaded into the central channel is limited to one type. In the present invention, each functional unit may be loaded with the same or similar type of sample, or each functional unit may be loaded with, for example, different types of samples.

[0023] In this specification, "sample" means a suspension containing cells or other organelles capable of spontaneous self-migration, or a suspension of chemical components added to induce any change in the observed physiological function. "Sample type" may refer to samples with different compositions and should therefore be examined individually. In this invention, different types of samples may be placed in separate functional units on the same device.

[0024] Typically, for example, the type or dosage of a drug may differ for each sample administered on the same device. Other scenarios include samples with different cell types, or samples with different concentrations or types of other chemical factors that define the cellular (or cancer) microenvironment.

[0025] In an advantageous embodiment of the present invention, the length of the working portions of the first flow path and the second flow path (the length of the portions of the flow paths that are interfaced and connected to each other via the microchannel array, that is, the width of the microchannel array) can be shortened compared to the prior art, which enables faster imaging, captures sites more rapidly, and thus can reduce photobleaching and ultraviolet exposure to the loaded cells. Additionally, this novel design allows simultaneous imaging on a single chip.

[0026] Due to the unique structure of the device, such as the size and shape of the microfluidic flow paths, it is possible to arrange 2 to 10 (depending on the embodiment), for example 3 to 5 or 6 to 10 functional units in a single microfluidic chip, for example to fit the size of a standard microscope slide. The surface area of the microscope tray holder is 85.5 mm×127.5 mm, or fits 85.5 mm×127.5 mm. Accordingly, the size range of the microfluidic chip can be defined between the standard microscope slide size of 26 mm×76 mm and the overall size of the tray which can incorporate a large slide with multiple functional units or incorporate a plurality of standard microscope slides arranged on an adapter frame having a total surface area of 85.5 mm×127.5 mm. The size and structure of the device, as well as the size and structure of components such as the flow paths and functional units, can be adjusted according to the size of experiments and microscope slides, for example, to fit the stage of a standard microscope and / or microscope scanner device.

[0027] When the functional units of a microfluidic device are arranged on a microplate instead of a microscope slide, for example, a microfluidic device having 1 to 384 functional units can be constructed. A further advantage of such a microplate approach is that in addition to pipetting, imaging can be further automated using, for example, robotic pipetting or a plate reader. Accordingly, the present invention can provide a novel method for constructing a microfluidic chip device that enables testing and / or imaging of a plurality of samples on a single chip.

[0028] By providing a plurality of, for example, two or more, or three or more functional units, parallel and simultaneous monitoring of an immune response (that is, observation of the migration of immune cells to cancer cells) can be achieved, for example, under 3 to 10 different conditions (different drug combinations or dosages). The size and number of the functional units can be determined according to the area of the chip, the area of the microscope stage, and / or the area of the scanning region of the microscope. Depending on the type of the drug, the drug can be mixed with one or both of an immune cell-containing medium and a cancer cell-containing medium before injection. When the device is used in other scenarios, a mixture or combination of a plurality of drugs can be added to the liquid before injection into the chambers on both sides.

[0029] By mounting a plurality of functional units in one device, different samples can be monitored simultaneously while monitoring the different samples, for example, without removing the slide carrying the device from a reader (e.g., a fluorescence microscope or a microscope slide scanner).

[0030] The working portion is defined as the entire flow channel section from both sides along the microfluidic array, i.e., the portion of the first and second flow channels that are in fluid communication with the microfluidic array. The length of the working portion of the flow channels in the device according to embodiments of the present invention can also be beneficial for imaging, because imaging a single unit reduces the time required for imaging, the number of shots taken, and the number of images to be stitched together. Assuming a wide objective lens is used for the reader, the entire working portion can be imaged in a single imaging shot, eliminating the need for image stitching. Alternatively, only specific sections can be selected and imaged.

[0031] The presented chamber and flow path structure allows the entire functional unit to be made at least 30%, preferably at least 50%, or more preferably at least 75% shorter than that of the prior art.

[0032] Shortening the length of the channels offers the obvious advantage of reducing the amount of sample required to inject into the device. This is particularly important when the number of available cells in the sample (e.g., isolated from a biopsy sample) is limited, but the cell concentration in the culture medium must be maintained equally for the device to function properly. The proposed configuration provides an environment in which the cell populations on both sides of the microfluidic array can properly interface with each other; that is, the cells are evenly distributed on both sides of the microfluidic array. The width of the microfluidic array, i.e., the length of the working portions of the first and second channels, can be 30 μm to 9000 μm, preferably 3000 μm to 7000 μm, and more preferably 4000 μm to 6000 μm.

[0033] According to one embodiment, the width of the microfluidic array can be 30 μm to 2000 μm, preferably 50 μm to 100 μm. This may be advantageous when the sample volumes are very small, but a high concentration ratio between cell volume and total volume is required by the assay.

[0034] Furthermore, in the provided device, where the channel geometry (first channel, second channel, and / or microfluidic array) provides channels with smaller volumes than those of the prior art, the volume or cavity requires less fluid to fill the channels, so the device can function with smaller samples. This is a clear advantage when the number of available or required cells, spheroids, or organoids is limited, or when the availability and cost of the drugs or other substances to be tested are limited.

[0035] Furthermore, the disclosed microfluidic device has a flow path geometry (specifically, a short flow path or at least a short working portion of the flow path) that can enable reliable filling of the first and / or second chambers by an automated pipette. Loading should be performed while preventing bubble formation in the flow path, which can be difficult to achieve when using an automated pipette. Because the flow paths of the prior art are long, if the viscosity is high, the pressure drop of the injected fluid becomes large along the flow path and may exceed the ability of the automated pipette to generate laminar flow during loading. As a result, bubbles may form during loading in the prior art device. However, the present invention enables laminar flow along the flow path and essentially prevents turbulence during injection, and therefore bubble formation can be essentially eliminated or at least reduced.

[0036] According to one embodiment, the surface area of ​​the chambers may vary depending on their intended loading volume. Chambers with smaller volumes may have smaller surface areas or radii, which typically correspond to an essentially circular cross-section with a radius of 200 μm to 400 μm, or match the outer radius of the loading device (e.g., pipette), in order to provide a tip seal when the cell suspension is injected. A proper seal is important to prevent bubble formation during injection. On the other hand, chambers with larger intended volumes may have larger surface areas to facilitate gas exchange on the chamber surface.

[0037] This device can provide a platform for predicting the efficacy of drugs intended for use in immunotherapy in individual patients with cancer and other diseases. It is intended to be used as an in vitro or ex vivo microfluidic platform for developing personalized strategies in the treatment of diseases where evaluating the immune response induced in individual patients is essential to identifying the optimal type, dosage, and combination of drugs before administering them to individual patients.

[0038] A device according to one embodiment of the present invention may include glass, PDMS, Flexdym polymer, polystyrene, polypropylene, polycarbonate, PMMA, ceramic, silicone, and / or thiol-ene. The material for the functional section of the flow channel can be produced from PDMS or a suitable derivative of PDMS that provides appropriate gas permeability. When considering a composite assembly of the device, other materials may be used (glass, Flexdym, thiol-ene, or any derivative thereof, COC, or any block copolymer having sufficiently low turbidity and high transparency).

[0039] As those skilled in the art will understand, the above description relating to various embodiments of the device can be flexibly applied to embodiments of methods for manufacturing the device, with necessary modifications, and vice versa. [Brief explanation of the drawing]

[0040] The drawings are provided to illustrate the disclosed embodiments and should not be construed as limiting the scope of use of the embodiments. The drawings are not to a specific scale.

[0041] [Figure 1] Figure 1(A) shows a prior art device, and Figure 1(B) is an inclined side view of an embodiment of the microfluidic chip device of the present invention on a microscope slide. [Figure 2] Figure 2 is an enlarged view of a portion of the inclined side view of the embodiment of the microfluidic chip device shown in Figure 1B. [Figure 3] Figure 3 is an enlarged view of a portion of the inclined side view of the embodiment of the microfluidic chip device shown in Figure 2. [Figure 4] Figure 4 shows an enlarged, inclined side view of a part of one embodiment of a microfluidic chip device. [Figure 5] Figure 5 is a top view of one embodiment of a microfluidic chip device on a microscope slide. [Figure 6] Figure 6 is a flowchart of a cell culture method according to one embodiment of the present invention. [Figure 7] Figure 7 is a flowchart of a method for manufacturing a device according to one embodiment of the present invention. [Modes for carrying out the invention]

[0042] The solutions will be described in more detail below with reference to several embodiments, but these should not be considered limiting.

[0043] In detailed descriptions, "chamber" and "circular well" may be used interchangeably. In detailed descriptions, "cell" may be used as an extended term to include cells, cell aggregates, spheroids, and organoids. In the context of detailed descriptions, the term "comprising" may be used as an open term, but also as a closed term, "consisting of."

[0044] Figure 1(A) shows a prior art device 001. This device comprises at least two primary chambers 002 connected by a primary channel 004. The device also comprises two sets of secondary chambers 006 connected by a secondary channel 008. On both sides of the primary channel 004, the primary channel 004 is connected to the secondary channel 008 via a primary microchannel array 010.

[0045] Device 001 also comprises a number of side containers or side chambers 012. The side chambers 012 are connected to secondary channels 008 by side channels 014 and secondary microchannel arrays 016. The side chambers 012 are positioned adjacent to the primary and secondary channels containing cells, and communicate with the secondary channels 008 via the secondary microchannel arrays 016. They serve as a fresh source of nutrients and are intended to replenish the gel matrix of the cell-containing channels or chambers, and also to prevent shrinkage during use.

[0046] In conventional devices, components 002-016 can be considered to form a single functional unit, which is suitable for studying one type of cell, one cell sample, or one type of cell interaction. Due to the space occupied by the functional unit, the number of functional units that can be used simultaneously on a single microscope slide, for example, is limited to only one.

[0047] Figure 1(B) shows a device 100 according to one embodiment of the present invention, which is placed on a microscope slide 200. The device 100 comprises one or more functional units 102, each of which comprises two first chambers 104 arranged to be in fluid contact by a first channel 106 and two second chambers 108 arranged to be in fluid contact by a second channel 110. A microfluidic array (not clearly visible in Figure 1) is arranged to connect the first and second channels.

[0048] The functional unit 102 can consist of two first chambers 104, a first flow path 106, two second chambers 108, a second flow path 110, and a microfluidic array.

[0049] The device 100 and the functional unit 102 may be provided without side chambers and side flow channels, as illustrated in relation to Figure 1(A).

[0050] The first chamber 104 and / or the second chamber 108 can be essentially cylindrical and have a circular base with a radius selected to provide a circular well for receiving fluid. The cylindrical wall can define the height of the chamber. The first chamber 104 and / or the second chamber 108 may also embody several different shapes, for example, the base can be rectangular. Along with the wall height, the size of the base shape, for example, may be selected to define the respective volumes of the first chamber 104 and / or the second chamber 108.

[0051] The device 100 in Figure 1(B) comprises three functional units 102. Other numbers of functional units 102 may also be employed. The size of the functional units 102, or the size of the flow path and / or chamber, may determine the number of functional units that may be employed for a particular use case scenario.

[0052] The device 100 is thought to include at least a substrate 112 that forms the base of the device 100. The substrate material may include, for example, silicone (polydimethylsiloxane (PDMS)), and the substrate material can define structures disposed on the device, namely chambers and flow channels. The thickness of the substrate can be 30 μm to 3.5 mm. The thickness of the substrate may be variable, i.e., the substrate may be locally thin. The thickness of the floor can be 0.03 mm to 3 mm, preferably 0.05 mm to 2 mm, more preferably 0.1 mm to 0.9 mm. The thickness of the roof can be 0.05 mm to 20 mm, preferably 0.1 mm to 3 mm, more preferably 0.11 mm to 2 mm. The thickness of the walls of the flow channels and / or chambers can be 10 μm to 50 μm.

[0053] During use, an immunocell suspension may be supplied to the first chamber 104, and cancer cells may be supplied to the second chamber. The immunocell suspension may contain at least immune cells in the medium. The immunocell suspension may further contain proteins and a mixture of other organic and inorganic compounds added for the purpose of mimicking the in vivo microenvironment of cancer cells.

[0054] A cancer cell suspension may include at least cancer cells suspended in a culture medium and a hydrogel-forming additive. For example, Matrigel or other viscous media supplemented with a gel-forming additive such as fibrinogen can be used.

[0055] Immune cells in an immunotherapy suspension before injection can be stained with a first cell tracking dye. Cancer cells in a cancer cell suspension can be stained with a second cell tracking dye. When immune cells interact with cancer cells, and as a result of this interaction the cancer cells die and lyse, the emission color of the fluorescent dye used to stain the cancer cells changes. This can be monitored under a fluorescence microscope, scanner, or other suitable alternative reading device.

[0056] Furthermore, this device can also be used to culture cells other than cancer cells and immune cells. The samples used may include, for example, materials that mimic the physiological phenomena under study. The samples may include, for example, drugs, hormones, antibodies, or cancer microenvironment-specific proteins.

[0057] Figure 2 shows a magnified portion of an inclined side view of an embodiment of the microfluidic chip device 100 of Figure 1. The first chamber 102 is partially shown together with the second chamber 108, the first channel 106, and the second channel 110. A microfluidic array 114 connecting the first channel 106 and the second channel 110 is also schematically shown.

[0058] The overall thickness of device 100 can be determined by the desired pipetting volumes, i.e., the volume of fluid delivered to chambers 104 and 108. The pipetting volumes can be used to determine the height and / or radius (or other dimensions if the chamber shape is not cylindrical) of the first and / or second chambers. The pipetting volumes can range from 1 μL to 1000 μL for each of the first and second chambers. The volume enclosed by the first and / or second chambers may be selected to essentially correspond to the pipetting volumes. The overall thickness of the microfluidic device can range from 130 μm to 2 cm. The overall height / thickness of device 100 may depend on the heights of chambers 104 and 108.

[0059] According to one embodiment, the defined range for the length of the channel can represent the type of experiment in question. In other words, since cells, spheroids, and organoids each have different diameters, the shape of the channel may vary depending on the diameter of the cells, spheroids, or organoids being injected. In versions of the device into which organoids or spheroids are injected, the depth and width of channel 110 can be twice the diameter of the spheroid. The sample volume for channel 110 may be equal to the total volume, and the volume of channel 106 may be equal to the sample volume. The sample volume can be injected directly into the channel without filling reservoir 106. The total volume of this section may be the volume of channel 106 plus two reservoirs, and may be filled with a cell culture suspension that does not contain cells.

[0060] The heights of chambers 104 and 108, along with their radii, determine the amount of fluid (cell culture) sample that can be loaded depending on the length of the experiment. The heights h1 and / or h2 of the first chamber 104 and the second chamber 108 are as follows: The height of chamber 104 can be 100 μm to 3 cm (if a cylindrical ring is attached to the base), and the height of chamber 108 may be the same as or different from chamber 104 within the above range.

[0061] In one embodiment, at least a portion of the device 100 may be covered with an essentially transparent material such as glass or a transparent polymer. Essentially all or most of the device 100, excluding the first chamber 104 and the second chamber 108, may be covered. Figure 2 shows region A of the device 100 that can be essentially covered. One or more (e.g., all) functional units 102 of the device 100 may be covered, and the covering is applied to at least the first channel 106, the second channel 110, and the microfluidic array 114.

[0062] Figure 2 shows the working portion of the flow path, which essentially corresponds to the width L1 of the microfluidic array. Therefore, the working portions of the first and second flow paths may refer to the portions of the flow path that are in fluid communication via the microfluidic array 114, and the length of the working portion of the flow path may be defined by the width L1 of the microfluidic array 114.

[0063] According to one embodiment, the functional unit may be configured such that the operating part includes a microfluidic array 114, a first channel 106 that is in fluid connection with the microfluidic array 114, and a second channel 110 that is in fluid connection with the microfluidic array 114. The portion of the first channel 106 that is in fluid connection with the microfluidic array 114 can be at least 75%, preferably at least 95%, and more preferably 100% of the total length of the first channel 106. The portion of the second channel 110 that is in fluid connection with the microfluidic array 114 can be at least 50%, preferably at least 75%, and more preferably at least 95% of the total length of the second channel 110.

[0064] The width L1 may be defined by the number of microchannels, their dimensions, and / or their spacing. The width L1 of the microchannel array may depend on the precision of the automated pipetting device used to load the chambers on both sides of the microchannel array. The width L1 of the microchannel array can be 30 μm to 9000 μm, preferably 3000 μm to 7000 μm, and more preferably 4000 μm to 6000 μm.

[0065] According to one embodiment, the width L1 of the microfluidic array can be 30 μm to 2000 μm, preferably 50 μm to 100 μm. This may be advantageous when the sample volume is very small, but a high concentration ratio between cell volume and total volume is required in the assay.

[0066] Figure 3 is an enlarged view of a portion of the inclined side view of an embodiment of the microfluidic chip device shown in Figure 2. The first channel 106, the second channel 110, and the microfluidic array 114 are shown.

[0067] The height h3 of the second channel can be 10 μm to 300 μm, preferably 170 μm to 190 μm, for example, 189 μm. The height of the first channel may be within the same range as the height of the second channel, or it may be equivalent. The heights of the first channel and / or the second channel may be optimized for better imaging (smaller depth of field) and for supplying more nutrients to cells, compared to the situation of the prior art. When the device is made of a single-layer slide such as PDMS, there is a trade-off between lowering the chamber height for better imaging and raising the chamber height for supplying more nutrients. If cylindrical sample holding rings are additionally attached to chambers 104 and 108, it is possible to achieve a thinner structure that enables better imaging by using larger sample holding components. When high magnification is required, the thickness of the polymer portion, i.e., the distance between the channel ceiling and the top surface of the substrate, should be selected to allow access for high-magnification microscope lenses with a much smaller depth of field.

[0068] The width of the first channel 106 can be 200 μm to 3000 μm, preferably 1190 μm. The first channel may be used for immune cells or types of assay cells that are large in volume and / or easy to reach (easier attainability). The width of the second channel 110 can be 100 μm to 1000 μm, preferably 490 μm. The second channel 110 may be used for cancer cells or types of assay cells that are small in volume and / or difficult to reach.

[0069] Figure 4 shows an enlarged inclined side view of a part of one embodiment of the microfluidic chip device 100. Specifically, Figures 4(A) and 4(B) illustrate a part of the microfluidic array 114 according to one embodiment of the present invention.

[0070] The microfluidic array 114 comprises a plurality of microfluidic channels 116. Each of the microfluidic channels 116 may have a height h4 of 5 μm to 20 μm, preferably 10 μm to 15 μm.

[0071] The microchannels 116 may be spaced apart by a distance d1 of 20 μm to 40 μm, preferably 27 μm to 37 μm, for example, 32 μm. One intended function of the spacing or distance between microchannels is to separate cells from each other when entering a microchannel and when exiting a microchannel on the other side. This prevents cells (e.g., cancer cells) from tilting onto adjacent microchannels, causing the cells to interact and block the microchannel at the exit.

[0072] The microchannel 116 can have a width w1 of 10 μm to 15 μm, preferably 11 μm to 13 μm, for example, 12 μm.

[0073] The length l1 of each individual microchannel 116 within the microfluidic array may be different. Several different lengths l1 can be used to achieve different spatial gradient scenarios for chemical signal transduction and migration distance. The length l1 of the microchannel 116 (or microfluidic array 114) can be 10 μm to 2000 μm, preferably 10 μm to 1000 μm. To select the length l1 of the microchannel (array) and the number of microchannels 116, the lengths of the first and / or second channels on both sides of the microfluidic array (or at least a portion of the lengths of the first and second channels that are in physical contact with the microfluidic array 114) can be taken into consideration. The lengths of the first and / or second channels may be determined by the sample volume(s). The sample volume(s) can be 1 μL to 1000 μL on both sides. The cell content of the cell suspension volume can be 0.1 μL to 149 μL, preferably 0.1 μL to 2.9 μL, more preferably 0.5 μL to 2 μL, and even more preferably 1 μL to 2 μL, and the number of microchannels 116 can be 3 to 250. The microchannels may be grouped into subgroups of unevenly distributed microchannels.

[0074] According to one embodiment, the optimal volume of the second channel, for example, the second channel on the cancer cell side, can be 1 μL to 5 μL including the cell suspension. If the second channel is filled with tumor spheroids or organoids, the channel volume can be in the range of 5 μL to 25 μL. The first channel side usually contains an immune cell suspension or other types of cells, and its volume is larger, with an optimal volume of 20 μL to 1000 μL, typically 50 μL to 200 μL.

[0075] The microchannels 116 of the microfluidic array 114 are driven by capillary forces and can be probed and filled with liquid / gel from both sides. Air pockets may remain within the microchannels. These air pockets usually disappear by dissolving in the culture medium and / or by being spontaneously extruded through the nanopores of the device's polymer matrix. This extrusion is facilitated by capillary forces that draw liquid into the microchannels from both sides. Another important requirement is that liquid does not enter the microchannels while filling them. This characteristic is influenced by the surface properties of the polymer used facing the internal cavities of the microchannels. By using material selection and modifying surface properties, the formation of permanent bubbles can be avoided and the removal of trapped air pockets inside the device can be facilitated.

[0076] According to the embodiments shown in Figures 1 to 4, the second flow path 110 is arranged to form two 90-degree angles. This is not mandatory. The second flow path 110 may be linear, or it may have any angle that provides a suitable distance between the second chambers 108, i.e., any angle that provides sufficient space for convenient and safe pipetting.

[0077] Figure 5 is a top view of one embodiment of a microfluidic chip device on a microscope slide. The surface area or radius of the chambers (first chamber and second chamber) may vary based on the radius of the pipette tip and the width of the microscope slide.

[0078] The surface areas of each of the first chambers 104 and each of the second chambers 108 can correspond to essentially circular cross-sections with radii of 0.5 mm to 10 mm. If chambers 104 and 108 are larger than this, it becomes difficult to fit them onto a single standard microscope slide without affecting structural stability or the ease of device assembly.

[0079] According to one embodiment, the surface area or radius of each of the second chambers 108 may be small enough to provide a seal at the pipette tip when a suspension of cancer cells or other types of cells is injected, typically corresponding to an essentially circular cross-section with a radius of 200 μm to 400 μm, or matching the outer radius of the loading device (e.g., pipette). A proper seal is crucial to prevent bubble formation during injection.

[0080] According to one embodiment, the surface area of ​​each of the first chambers 104 may be larger than the surface area of ​​each of the second chambers 108. Each of the first chambers 104 retains excess liquid for the purpose of both supplying nutrients to cells (typically immune cells) injected into the channel 106 before filling the reservoir, and for the purpose of mitigating the effects of evaporation. A typical range of surface area of ​​the first chambers 104 can correspond to an essentially circular cross-section having a radius of 3 mm to 5 mm.

[0081] The larger the surface area of ​​the pipetting area of ​​the first chamber 104, the better it can function as an open air container for the culture medium containing cellular nutrients in the immunotherapy cell suspension, allowing for better gas exchange between the liquid and the incubator atmosphere.

[0082] The smaller the pipetting area of ​​the second chamber 108, the more the evaporation of the gel sample injected into the narrow second channel can be limited, and thus the contraction of the channel contents can be prevented. The radius of the injection port can be selected according to the physical properties of the suspension being loaded and the type of cells (if required to be in a gel matrix) to achieve better viability, cell number, and the need for gas exchange of the suspension.

[0083] When the microfluidic device 100 is arranged on a microplate, similar dimensions can be used for the individual functional units 102. The functional units 102 may be provided independently for each well of the microplate. Alternatively, a microplate-sized flat surface, such as glass, may be used, and each of the functional units 102 may be arranged on the flat surface according to the method described in Figure 7 and its description in this application. When a flat surface is used, the position of each functional unit 102 may be positioned to coincide with the well positions on a microplate having a desired number of wells. For example, if the microfluidic device 100 is arranged to have 96 functional units 102, the positions of the functional units 102 may be positioned to coincide with the well positions of a microplate with 96 wells or 384 wells. This allows a microplate reader to obtain, for example, one, two, or four reading positions from each of the functional units 102. Regardless of how the functional units 102 are assembled on the microplate, the resulting solution enables repeated automated analysis of all functional units 102, each defined as a separate well or a corresponding well location.

[0084] Figure 6 is a flowchart of a cell culture method according to one embodiment of the present invention. First cells are provided for cell culture, for example, from human peripheral blood mononuclear cells (120). The first cells are prepared and a first sample is formed from the first cells (122). Second cells are provided for cell culture, for example, from cancer cells or organoids (130). The second cells are prepared and a second sample is formed from the second cells (132). The second sample is injected into a second channel 110 on device 100 (134). The device into which the second sample has been injected is incubated in a cell culture incubator for, for example, 30 minutes (134). Thereafter, the first sample is injected into a first channel 106 on device 100 (124). Nutrient medium is added to the first chamber 104 to ensure the viability and osmotic pressure of the first cells in the first sample (126). After both channels 106 and 110 have received their samples, the device with the samples is incubated for, for example, 12 to 72 hours (138). After incubation, the first cells from the first sample and the second cells from the second sample move into the microfluidic array 114 of the operating part of the functional unit 102, and the samples are imaged (140).

[0085] According to exemplary embodiments, the sample injected into the second channel 110 can be prepared from a tumor tissue sample or a corresponding sample such as an organoid. The sample under study can be obtained by biopsy, perioperatively, or by other means. If the sample is a tumor sample, the sample may be taken from a region adjacent to the center of the tumor to ensure the presence of the tissue cells of interest or cancer-associated fibroblasts. A key fragment of the sample is preferably 1 mm. 3 ~2mm 3The tissue may be separated and then finely chopped. The sample pieces may be finely chopped in ice-cold Hanks equilibrium salt solution (HBSS; supplied with 100 U / ml penicillin, 100 μg / ml streptomycin, and 250 ng / ml fungizone). The sample pieces can then be centrifuged at 4°C and 1000 rpm (200 × g) for 5 minutes. The supernatant may then be discarded, and fresh HBSS buffer may be added before repeating the centrifugation step. The tissue pellet can then be suspended in 5 ml of HBSS buffer containing 1 mg / ml collagenase type I from Clostridium histolyticum and placed on a rocker platform at 37°C for 2 hours. The tube may then be centrifuged, the supernatant discarded, replaced with fresh HBSS buffer, and then centrifuged again. The processed sample can be suspended in HBSS buffer, filtered through a 100 μm cell strainer, and the passing single cells can be collected and centrifuged. You may discard the supernatant and suspend the cell pellet in DMEM / F-12.

[0086] According to exemplary embodiments, a cell pellet from cancer cells or organoids may be stained, for example, with Celltrace Far Red and suspended in a solution that is liquid at room temperature in a culture medium. This may be injected into channel 110 and then used to form a hydrogel ECM. This solution may be prepared from the following components at the following concentrations: 2.4 mg / ml Myogel, 0.5 mg / ml fibrinogen, 0.3 U / ml thrombin, and 33.3 μg / ml aprotinin. These reagents may be diluted in DMEM / F12 medium containing 10% patient serum and an immune checkpoint inhibitor. For example, PD-1 inhibitors, fluorescent proteins and biosensors for super-resolution microscopy imaging, or genetically encoded optogenetic tools for manipulating biological processes using light may be added. Possible alternatives to myogel include Lymphogel, ECMgel (Sigma), Cultrex® BME (Amsbio), Geltrex® (Gibco Life Technologies), and ECMatrix® (Millipore). These products are homogenates of mouse tumor tissue and have a different composition from human TMEM, thus having the same drawbacks in human studies. Additional components may then be added in amounts that result in a number and concentration of cells in the sample suitable for a given microfluidic dimension and volume set to maximize the number of cells introduced into the fluidic cavity. The additional components can prevent the formation of cell clusters. If organoids are used instead of cancer cells, the organoids can be applied at a concentration that allows them to be introduced into the microfluidic without forming aggregates.

[0087] According to exemplary embodiments, the sample injected into the first channel 106 can be prepared from human peripheral blood mononuclear cells (MNCs) or a corresponding sample. The sample may be isolated from target tissue or an in vitro cell culture. When human peripheral blood MNCs are used, they may be isolated from the pia (buffy coat) of cancer patients. MNCs may be isolated via density gradient spectroscopy. Peripheral blood MNCs consist of adaptive and innate immune cells (T cells, B cells, NK cells, monocytes, and dendritic cells) individually or as mixtures of any combination thereof. T cells may be isolated and further processed according to a CAR-T cell therapy protocol. They may then be used alone or in combination with other immune cells for in vitro evaluation of efficacy as monotherapy or combination therapy. The process of incorporating genes encoding chimeric antigen receptors (CARs) into T cells can be carried out according to any protocol defined as part of any CAR-T cell therapy. Serum from cancer patients can be prepared by coagulating it at room temperature for 30 minutes and then centrifuging it at 2000 rpm for 10 minutes in a refrigerated centrifuge at 4°C. This serum may be added at a concentration of 10% by volume when preparing the immunocellular suspension. Immunostimulants and immune checkpoint inhibitors, or fluorescent proteins and biosensors for super-resolution microscopy imaging, or genetically encoded optogenetic tools for manipulating biological processes using light may be added. Other components may be added in amounts that result in a number and concentration of cells in the sample appropriate for a given microfluidic dimension and volume set to maximize the number of cells introduced into the microfluidic cavity. Additional components can prevent the formation of cell clusters and aggregates.

[0088] According to an exemplary embodiment, a sample containing a cancer cell suspension can be loaded into the second channel 110. The tip is then placed in the incubator for 30 minutes to form an ECM hydrogel. Next, a sample containing an immunocellular suspension can be loaded into the first channel 106. Following the direct injection of the cell suspension into the first channel 106, additional buffer can be loaded into the first chamber 104 to supply nutrients and prevent drying of the first channel 106 during culture.

[0089] According to one embodiment, the device 100 may be placed in a cell culture incubator after both the channel 110 and the channel 106 are loaded. Within 12 hours of placing the device 100 in the cell culture incubator, the microfluidic array 114 between the parallel sections of the first channel 106 and the second channel 110, which form the working part of the functional unit 102, is primed, and air bubbles trapped in the microfluidic array 116 can be removed. After incubation, the device 100 may be imaged multiple times over the next 36 hours using a fluorescence microscope. During this time, an immune response to cancer cells is formed, and immune cells migrate towards the cancer cells through the microfluidic array 116. Upon reaching the cancer cells, the cancer cells may be neutralized because immune checkpoints are blocked. While the device 100 is being imaged, the number of immune cells passing through the microfluidic array 114 is counted, serving as a direct indicator of the immune response. Cancer cells neutralized by immune cells can be observed by a change in color in their fluorescence. The number of cancer cells attacked and killed by immune cells can be a direct indicator of the effectiveness of drug therapy or combination drug therapy. As will be apparent to those skilled in the art, the procedure described above can be repeated in parallel in multiple functional units, thereby enabling simultaneous dual sampling, reference sampling, and sampling with different doses, different cells, or different drugs.

[0090] According to one embodiment, multiple drugs and / or drug candidates, or combinations of drugs and / or drug candidates, can be used. These may include, but are not limited to, the drugs and drug candidates listed below, and the drugs and drug candidates listed in Tables 1 to 4 below.

[0091] Table 1: Examples of drugs and drug candidates that have inhibitory function against immune checkpoint targets

[0092] [Table 1]

[0093] Table 2: Drugs and drug candidates that have inhibitory functions other than targeting immune checkpoints.

[0094] [Table 2]

[0095] Table 3: Existing immune checkpoint inhibitors

[0096] [Table 3]

[0097] Table 4: Novel immune checkpoint inhibitor molecules and other inhibitory targets

[0098] [Table 4]

[0099] In addition to the drugs and drug candidates listed in Tables 1 to 4, immune checkpoint inhibitors and drug candidates developed in China, such as Tuoyi (toripalimab), Tyvyt (Sintilimab), Tislelizumab, Camrelizumab, AK105, CS1001, CS1003, zimberelimab, HLX-10, KN046, and SHR-1316, may also be used. Other humanized IgG4 anti-PD-1 monoclonal antibody drugs and drug candidates, such as Spartalizumab (PDR001), Dostarlimab, TSR042, MGA012, Sasanlimab (PF-06801591), Budigalimab (ABBV-181), and BI754091, may also be used. In addition, the subcutaneously administered PD-L1 nanobody Envafolimab (KN035), the fully human IgG1 PD-L1 monoclonal antibody drug or drug candidate Cosibelimab (CK-301), the TGFβRII extracellular domain fused to the bifunctional fusion protein, the human IgG1 PD-L1 monoclonal antibody drug or drug candidate Bintrafusp alfa (M7824), and / or small molecule inhibitors of VISTA, PD-L1, and PD-L2 CA-170 may also be used.

[0100] Figure 7 is a flowchart of a method for manufacturing a device according to one embodiment of the present invention. A substrate is provided for manufacturing the device (150). The substrate is cast to form the necessary fluid communications within the device (152). Fluid communications are provided between the first chamber 104 and the first channel 106, between the second chamber 108 and the second channel 110, between the microfluidic array 114 and the first channel 106, and between the microfluidic array 114 and the second channel 110 (154). Finally, the substrate is cured to obtain a functional device 100 (156).

[0101] The method for fabricating the microfluidic device 100 can be based on standard soft lithography as described in the prior art. The substrate thickness can be 30 μm to 3.5 mm. If the substrate thickness is locally reduced, the vertical distance over which bubbles escape from the microchannel is shortened, which may increase gas permeability.

[0102] Since the microfluidic array 114 is loaded from both the first channel 106 side and the second channel 110 side, bubbles are forced to remain within the microfluidic array 114. Therefore, bubbles trapped in the microfluidic channel 116 before and during sample loading remain within the microfluidic array 114. Increased gas permeability allows bubbles to escape more quickly, which in turn allows for faster priming of the microfluidic array 114.

[0103] To provide an appropriate level of gas permeability for cell culture and to facilitate the removal of bubbles from the microchannels during priming of device 100, it is preferable to use PDMS or other materials with equivalent gas permeability for the functional parts of device 100 for cell culture and cell migration. PDMS structural components can be shaped to the desired form by casting onto a mold and then thermocuring the material after mixing with a crosslinking agent. Photocurable PDMS can also be used, and photocuring can be applied after casting or as part of an additive manufacturing (3D printing) process. Furthermore, roll-to-roll nanoimprinting can be used in both thermocuring and photocuring processes, or a combination thereof.

[0104] In addition to PDMS, other thermoplastic materials such as polystyrene, polymethyl methacrylate (PMMA), poly(ethylene glycol) diacrylate (PEGDA), cyclic olefin copolymer (COC), and cyclic olefin polymer (COP), or any other thermoplastic plastic, or any combination of these materials, can be used alone or in combination to form both the structural and functional parts of device 100. Using these materials, the channels and support structures of the main chip structure can be shaped as intended by microinjection molding or hot embossing. Roll-to-roll embossing and roll-to-roll imprinting are alternative methods for mass production.

[0105] Molds for casting or molding can be manufactured by additive manufacturing, CNC micromilling, electroplating, or lithography, and these methods may include various methods for etching or adding material to the molding tool that forms the microfluidic structure to be replicated.

[0106] Many modifications of this application will be suggested to those skilled in the art in light of the foregoing description. Such obvious modifications fall within the fully intended scope of the appended claims. Some embodiments of the present invention are described in the following sections [1]-

[16] . [Item 1] A microfluidic device (100), - Gas permeable substrate and, - comprising one or more functional units (102), Each functional unit (102) is: -Two first chambers (104) arranged to be in fluid communication through a first flow path (106), -Two second chambers (108) arranged to be in fluid communication through a second flow path (110), - comprising a microfluidic array (114) arranged to connect the first flow path (106) and the second flow path (110), - The microfluidic array (114), the portion of the first channel (106) that is in fluid communication with the microfluidic array (114), and the portion of the second channel (110) that is in fluid communication with the microfluidic array (114) form the operating parts of each functional unit (102). Microfluidic device (100). [Item 2] The microfluidic device (100) described in item 1, wherein each functional unit (102) is a separate unit, is not in fluid communication with any other functional unit (102), and is preferably configured to be loaded independently. [Item 3] A microfluidic device (100) according to item 1 or item 2, wherein the width (L1) of the microfluidic array is in the range of 30 μm to 9000 μm, preferably 3000 μm to 7000 μm, and more preferably 4000 μm to 6000 μm. [Item 4] A microfluidic device (100) according to any one of items 1 to 3, wherein the width (L1) of the microfluidic array is in the range of 30 μm to 2000 μm, preferably 50 μm to 100 μm. [Item 5] The microfluidic device (100) is a microfluidic device (100) described in any one of items 1 to 4, which is placed on a slide for microscopic examination. [Item 6] The microfluidic device (100) is a microfluidic device (100) described in any one of items 1 to 5, which is placed on a microplate. [Item 7] A microfluidic device (100) as described in any one of items 1 to 6, wherein at least a portion of the microfluidic device (100), or essentially all or most of the microfluidic device (100) excluding the first chamber (104) and the second chamber (108) as optionally, is covered with a transparent material. [Item 8] Each of the first chambers (104) has a height of 100 μm to 3 cm and an essentially circular cross-section having a radius of 0.5 mm to 10 mm, preferably 3 mm to 5 mm, or any other two-dimensional shape having an area of ​​0.7 mm² to 314 mm², preferably 28 mm² to 78.5 mm², as described in any one of items 1 to 7. [Item 9] Each of the second chambers (108) has a height of 100 μm to 3 cm, A microfluidic device (100) according to any one of items 1 to 8, having a surface area corresponding to an essentially circular cross-section, with a radius of 200 μm to 400 μm, or a radius corresponding to the outer diameter of a loading device used to load each of the second chambers (108). [Item 10] A microfluidic device (100) according to any one of items 1 to 9, wherein the height of the first channel (106) is 10 μm to 300 μm, preferably 170 μm to 190 μm, for example 189 μm; the width of the first channel (106) is 200 μm to 3000 μm, preferably 1190 μm; the height of the second channel (110) is 10 μm to 300 μm, preferably 170 μm to 190 μm, for example 189 μm; the width of the second channel (110) is 100 μm to 1000 μm, preferably 490 μm; the length of the first channel (106) is at least the length of the microfluidic array (114); and the length of the second channel (110) is at least the length of the microfluidic array (114). [Item 11] Each of the microchannels (116) within the microchannel array (114) is A height (h4) of -5μm to 20μm, preferably 10μm to 15μm, -10μm to 15μm, preferably 11μm to 13μm in width (w1), -100 μm to 2000 μm, preferably 100 μm to 1000 μm in length (l1), The distance (d1) between the microchannels (116) is -20 μm to 40 μm, preferably 27 μm to 37 μm, A microfluidic device (100) as described in any one of items 1 through 10. [Item 12] The microfluidic device (100) is a microfluidic device (100) according to any one of items 1 to 11, comprising 1 to 384 functional units (102). [Item 13] The microfluidic device (100) described in any one of items 1 to 12, wherein the microfluidic device (100) includes PDMS. [Item 14] A method for culturing cells, providing a microfluidic device (100) as described in any one of items 1 to 13, To provide at least immune cells to at least one first chamber (104), To provide at least cancer cells to at least one second chamber (108), Incubating the microfluidic device (100), Observe the migration of immune cells to the cancer cells and count the immune cells and / or the cancer cells, Methods that include... [Item 15] The method according to item 14, comprising providing at least immune cells to at least one first chamber (104) and / or providing at least cancer cells to at least one second chamber (108) by one or more automated pipettes. [Item 16] A method for manufacturing a microfluidic chip, - To provide a substrate, - The thickness of the substrate is limited to 30 μm to 3.5 mm, and the substrate is processed to define one or more functional units (102). - To cure the substrate, Includes, Each functional unit (102) -Two first chambers (104) arranged to be in fluid communication through a first flow path (106), -Two second chambers (108) arranged to be in fluid communication through a second flow path (110), - A microfluidic array (114) arranged to connect the first flow path (106) and the second flow path (110), - comprising an operating part arranged to connect the portion of the first channel (106) that is in fluid communication with the microfluidic array (114) and the portion of the second channel (110) that is in fluid communication with the microfluidic array (114), A method for manufacturing microfluidic chips.

Claims

1. A cell culture method, A microfluidic device (100), - A gas-permeable substrate, - comprising one or more functional units (102), Each functional unit (102) is: - Two first chambers (104) are arranged to be in fluid communication through a first flow path (106), - Two second chambers (108) arranged to be in fluid communication through a second flow path (110), - comprising a microfluidic array (114) arranged to connect the first fluid channel (106) and the second fluid channel (110), - To provide a microfluidic device (100) in which the microfluidic array (114), the portion of the first channel (106) that is in fluid communication with the microfluidic array (114), and the portion of the second channel (110) that is in fluid communication with the microfluidic array (114) form the operating parts of each functional unit (102), To provide at least immune cells to at least one first chamber (104), To provide at least cancer cells to at least one second chamber (108), Incubating the microfluidic device (100), While observing the migration of the immune cells to the cancer cells, the immune cells and / or the cancer cells are counted. A cell culture method, including the following.

2. A cell culture method according to claim 1, comprising providing at least immune cells to at least one first chamber (104) and / or providing at least cancer cells to at least one second chamber (108) by one or more automated pipettes.

Images