Microfluidic device capable of simulating tissue or organs using cells

Abstract

The present invention relates to a microfluidic device capable of simulating tissues or organs using cells.

Description

Microfluidic devices that can mimic tissues or organs using cells

[0001] The present invention relates to a microfluidic device capable of simulating tissues or organs using cells.

[0002] Inflammation, a classic response of the host immune system to invading pathogens or endogenous signals associated with tissue repair, can lead to a variety of diseases. Acute inflammation occurs as an immediate response to injury and is characterized by biochemical reactions, including the migration of white blood cells, such as neutrophils and monocytes / macrophages, from the bloodstream to the damaged site / tissue. Chronic inflammation relies on complex biochemical reactions as well as innate and adaptive immune cells. Long-term stressors, such as infection, physiological stress, nutritional imbalances, and environmental factors, can activate inflammatory cells through the production of cytokines and chemokines, leading to chronic inflammation.

[0003] Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory lung disease characterized by persistent airflow limitation, including emphysema and / or chronic bronchitis. It is a common, irreversible disease, and associated with high mortality and disability worldwide. The primary etiology of COPD is exposure to irritating gases or particulate matter, such as pollutants or tobacco smoke, combined with genetic, developmental, and social factors. The pathogenesis of COPD is complex and heterogeneous, leading to airway disease, emphysema, mucus protein synthesis, and inflammation. Inflammatory cell migration and airway remodeling, mediated by neutrophils, CD8 T lymphocytes, and activated macrophages, are hallmarks of COPD onset and progression. Neutrophil transendothelial migration (NTEM) in lung tissue is associated with COPD severity. This process involves inflammatory cytokines and chemokines, such as leukotriene B4 (LTB4), interleukin-8 (IL-8, CXCL8), complement component 5a (C5a), tumor necrosis factor-α (TNF-α), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and other mediators in the lungs. Several attempts have been made to develop COPD drugs that inhibit the action of cytokines or chemokines or block their receptors. Glucocorticoids and bronchodilators are the mainstay of COPD treatment, but they are accompanied by side effects such as infection and immunosuppression.

[0004] The development of new drugs for human diseases relies on having appropriate animal models that mimic these diseases. Organ-on-a-chip (OoC) devices offer a way to at least partially circumvent the use of animal models. To observe the spontaneous migration of neutrophils from a single drop of blood from a sepsis patient, we used a microfluidic device designed with channels and mazes to analyze specific patterns of neutrophil motility. Previous studies demonstrated primary neutrophil migration from samples of sepsis and COPD patients on chips with and without an endothelial barrier, respectively. The inhibition of neutrophil transepithelial migration by the CXCR2 antagonist MK-7123 was investigated in a virus-induced asthma exacerbation model using an airway lung chip. Neutrophil migration induced by inflammatory mediators and various inflammatory diseases has been observed using various approaches, including two-dimensional migration assays, microfluidic chips, zebrafish, and mouse models. Additionally, there are no reports on the effect of pharmacological agents on neutrophil transendothelial migration using OoC with a full 3D interface.

[0005] In this study, we developed a novel 3D inflammation-on-a-chip (IoC) model and used it to evaluate the pharmacological effects of a CXCR2 antagonist. Our results demonstrated that MK-7123 significantly inhibited poly(I:C)-induced inflammation and NTEM in samples from COPD patients. This IoC model is the first to compare NTEM responses in plasma samples from COPD patients with those from healthy donors. Our 3D IoC model strongly mimics the COPD-induced inflammatory environment. It possesses a complete 3D interface between the endothelial barrier and the extracellular matrix (ECM) and a gradient of chemoattractants.

[0006] The purpose of the present invention is to provide a microfluidic device capable of simulating tissues or organs using cells in order to solve the above problems.

[0007] The present invention provides a microfluidic device, characterized in that a gel channel (Gel channel, 150) having a first height (H'2) is arranged in parallel with a common channel (Common channel, 120) having a second height (H1) on one side thereof, and a comparative channel (Comparative channel, 130) having a second height (H1) on another side thereof, and ports (110, 140) capable of accommodating liquid and allowing liquid injection and discharge are provided at both ends of the gel channel, the common channel, and the comparative channel, and the common channel (120) and the comparative channel (130) are distinguished by a substrate injected into the gel channel due to a height difference (H2) between the first height (H'2) and the second height (H1).

[0008] In one aspect, the present invention provides a microfluidic device capable of simultaneously testing an experimental group and a control group with two comparison channels.

[0009] In one aspect, the present invention provides a microfluidic device that forms a cavity structure between a gel channel and a common channel and a comparison channel, which is not separated by a fixed structure such as a phase guide, a pillar, a partition wall or a membrane.

[0010] In one aspect, the present invention can provide a microfluidic device in which a fixed structure such as a phase guide, pillar, partition or membrane is formed in a part or the entirety of each channel as needed between a gel channel and a common channel and a comparison channel.

[0011] The above port (110, 140) has a chamber having an inlet or an outlet, and the gel channel is in a straight line and has a port (110) at both ends, and the port (110) includes a chamber (201) having an inlet (202) or an outlet (202). In addition, the common channel (120) and the comparison channel (130) have ports (140) at both ends, and have a media inlet (204) and a media chamber (203).

[0012] In one aspect, the present invention provides a microfluidic device in which the first height (H'2) of the gel channel is higher than the second height (H1). Here, the height difference is preferably 1.5 to 3 times higher than the second height, and most preferably 1.7 to 2.8 times. In some embodiments, the second height (H1) is preferably in the range of 60 to 250 μm, and the first height (H'2) is preferably in the range of 60 to 260 μm higher than the second height (H1). That is, a first height in the range of 120 to 510 μm represents a preferred height range.

[0013] In one aspect, the present invention provides a microfluidic device in which the width of the gel channel is equal to or wider than the widths of the common channel and the comparison channel. Preferably, the gel channel width (D1) is 1.1 to 1.5 times the width of the common channel and the comparison channel (D2), and more preferably, 1.1 to 1.3 times the width of the common channel and the comparison channel (D2). In one embodiment, the width (D1) of the gel channel is preferably no more than 1 mm.

[0014] In another aspect, the above gel channel may be capable of allowing diffusion movement of substances injected into the common channel and the comparison channel.

[0015] In one aspect, the present invention provides a microfluidic device characterized in that a gel channel is filled with a matrix. The matrix is ​​preferably a hydrogel, and more preferably, the hydrogel is at least one selected from the group consisting of collagen, fibrin, alginate, gelatin, laminin, agarose, fibronectin, polyacrylamide, polyanhydride, hyaluronic acid, and polypeptide.

[0016] In another embodiment, the present invention provides an organ-on-a-chip comprising the configuration of the microfluidic device.

[0017] In another aspect, the present invention provides a method for measuring cell movement using a microfluidic device, comprising: 1) forming a matrix in a gel channel; 2) injecting vascular cells into one side selected from a common channel and a comparative channel to form a blood vessel wall; 3) injecting cells capable of movement into the side of 2) where the blood vessel wall has been formed; and 4) injecting a substance capable of inducing cell movement into the side where the blood vessel wall has not been formed; wherein the gel channel is capable of allowing diffusion movement of the substance injected into the common channel and the comparative channel.

[0018] The above vascular cells may be vascular endothelial cells.

[0019] The above blood vessel wall may be formed on at least one selected from the group consisting of a substrate surface, an upper inner side of the channel, an inner side of the channel, and an inner lower side of the channel.

[0020] The above-mentioned mobile cells may be one or more selected from the group consisting of neutrophils, monocytes, macrophages, and cancer cells.

[0021] The substance capable of inducing the above cell migration may be an inflammatory factor or a cancer metastasis inducing factor.

[0022] The above comparison channel may be a group in which conditions selected from a group consisting of an untreated group, a group treated with a substance that induces cell migration, a group treated with a substance that inhibits cell migration, and a group treated simultaneously with a substance that induces and inhibits cell migration can be compared.

[0023] The microfluidic device of the present invention may provide a suitable model for evaluating drug efficacy in inflammatory diseases whose pathogenesis and progression are mediated by neutrophils, and may strongly mimic the inflammatory COPD microenvironment and have the potential to investigate the severity of COPD.

[0024] Additionally, the microfluidic device of the present invention can also be used for basic research on NTEM and new drug development.

[0025] Figure 1 shows a perspective view (100) of a microfluidic device according to one embodiment of the present invention.

[0026] FIG. 2 shows a layout diagram of a gel channel, a common channel, and a comparison channel provided inside a microfluidic device according to one embodiment of the present invention.

[0027] FIG. 3 is a side view showing a cut-away portion of a microfluidic device according to one embodiment of the present invention.

[0028] FIG. 4 is a side view showing the difference between the first height (H2) and the second height (H1) of the microfluidic device according to one embodiment of the present invention, in which the gel channel (150), the common channel (120), and the comparison channel (130) are arranged without a fixed structure.

[0029] Figure 5 illustrates the process of fabricating a mold for a microfluidic device using a 3D printer according to one embodiment of the present invention. (a) The overall process used to create a 3D structured PDMS using a 3D printed mold. (b) The process used after extracting the PDMS from the mold. (c) A side view of the chip structure.

[0030] Figure 6 illustrates an example of a process for injecting an ECM mixture into a microfluidic device by pushing in one direction and pulling in the other direction.

[0031] Figure 7 illustrates a bidirectional method as an example of a process for injecting an ECM mixture into a microfluidic device.

[0032] Figure 8 illustrates a comparative inflammation-on-a-chip (IoC) model for measuring neutrophil transendothelial migration (NTEM). (a) A single device can be used to measure neutrophil transendothelial migration in plasma samples from healthy subjects and COPD patients, allowing for comparative analysis. (b) A schematic diagram of neutrophils and their representative surface receptors is shown. Neutrophils express CD11b (integrin alpha M), complement component 5a receptor (C5aR), leukotriene B4 receptor (LTB4R), CXCR1 (IL8RA), CXCR2 (IL8RB), and formyl peptide receptors 1 and 2 (FPR1 and FPR2).

[0033] Figure 9 illustrates the fabrication and preparation of a comparative inflammation-on-a-chip. (a) A diagram of the channel structure of the chip is shown. Two comparison channels are adjacent to the gel and common channels, allowing for comparison of samples under identical conditions and separated by 9.3 mm to eliminate interference. (b) A schematic diagram of a single module of the chip with representative cross-sections including the media chamber, media inlet, and migration region is shown. (c) A 3D-printed mold structure of the migration region is shown. The total channel width in the microfluidic device according to one embodiment of the present invention was 2 mm. The gel channel was 750 μm, and the common and comparison channels on both sides were 625 μm wide. The layer thickness of the 3D printer was 32 μm, and the optimal height was determined by adjusting H1 and H2. (d) The success rate (%) of extracellular matrix (ECM) restriction for each structure height condition is shown. For the neutrophil migration assay, a condition where both H1 and H2 were 160 μm (indicated by the red dotted line) was adopted. (e) Microscopic images showing the restricted ECM gel. (f) Top, bottom, and front images showing each channel of the IoC in different colors (red, common channel; yellow, comparison channel 1; green, comparison channel 2). (g) FITC-dextran diffusion analysis in inflammation-on-a-chip with and without endothelial barrier, with fluorescence intensity monitored for 5 hours (graph). Fluorescence images are shown at 2 hours (g-1) and 4 hours (g-2) with and without endothelial barrier, respectively.

[0034] Figure 10 depicts a 3D printing mold structure of a microfluidic device according to one embodiment of the present invention. (a) A schematic diagram of the upper mold is shown, detailing the size and names of the structures. (b) A schematic diagram of the lower mold is shown, detailing the size and names of the structures.

[0035] Figure 11 illustrates the optimization of post-curing conditions for a 3D printed mold of a microfluidic device according to one embodiment of the present invention. (a) A schematic diagram of the structural pattern used in the post-curing test is shown. The pattern line width was fixed at 512 μm, and the height and distance were adjusted. (b) Representative microscopic images clearly showing the molded pattern and the torn pattern as a result of controlling PDMS curing are shown. (c) Post-curing was optimized through comprehensive UV treatment and heat treatment. Heat treatment at 80°C for more than 24 hours was the optimal condition required to prevent PDMS curing inhibition in the 3D printed mold.

[0036] Figure 12 shows the expression of surface markers in differentiated HL-60 neutrophil-like cells. (a) Gating strategy using flow cytometry is shown. (b) Viability of control (PBS) and differentiated (10 μM ATRA, 500 μM dbcAMP, or 1.3% DMSO) HL-60 cells was measured. (c) Histograms of each surface marker under each differentiation condition are shown. (d) Normalized expression levels of receptors under each differentiation condition are shown. Expression levels of each receptor were normalized to those induced by 1.3% DMSO.

[0037] Figure 13 shows the results of a comparative analysis of dHL-60 cell migration. (a) The experimental setup for dHL-60 cell migration according to chemoattractant treatment is shown. (b) Representative fluorescence images of migrated cells are shown; the migration level is different depending on the media alone or the combination of IL-8, fMLP, C5a, LTB4 alone and these molecules (Combi). (c) The results of a comparison of the number of migrated dHL-60 cells under chemoattractant treatment and media treatment conditions are shown. (d) The experimental setup for determining the inhibitory effect of chemoattractant receptor blocker (CRB) on dHL-60 cell migration is shown. (e) Representative fluorescence images of migrated dHL-60 cells under control and CRB-treated conditions are shown. Migration levels were dependent on individual chemokines (IL-8, fMLP, C5a, and LTB4) and receptor blockers (anti-CXCR2, Boc-MLF, DF2593A, and LY223982). (f) Comparison of the number of migrated dHL-60 cells between control and CRB-treated conditions is shown (* = p < 0.05, ** = p < 0.01).

[0038] Figure 14 shows a comparative assay of dHL-60 cell transendothelial migration using poly(I:C)-conditioned medium (CM). (a) Schematic diagram of Calu-3 cell stimulation by poly(I:C). (b) Quantification of cytokines and chemokines in Calu-3 supernatants with or without poly(I:C) stimulation using ELISA. (c) Experimental setup for poly(I:C) CM-induced dHL-60 cell transendothelial migration. (d) Representative images (3D, maximum projection) of dHL-60 cell transendothelial migration induced by the untreated (NT) group and poly(I:C) CM group using confocal microscopy. (e) Comparison of the number of migrated dHL-60 cells between the NT group and the poly(I:C) group. (f) Experimental setup for drug efficacy assay on dHL-60 cell transendothelial migration. (g) Representative confocal microscopy images showing poly(I:C) CM-induced dHL-60 cell transendothelial migration in the presence of MK-7123 and control. (h) The number of migrated dHL-60 cells in the control and drug-treated groups (MK-7123, AZD5069, and SB225002) is shown (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

[0039] Figure 15 shows the expression pattern of adhesion proteins in HUVECs according to TNF-α treatment and the results of analysis of migration patterns according to adhesion protein inhibitor treatment. (a) Shows the gating strategy of HUVECs using flow cytometry. (b) Shows the histogram of each surface adhesion protein according to the TNF-α treatment time. (c) Shows the normalized expression level of adhesion proteins according to the TNF-α treatment time. The expression level of each adhesion protein was normalized to that induced by 2 hours. (d) Shows the fluorescence image of ICAM-1 (primary anti-ICAM-1, abcam, cat no., ab109361; secondary anti-rabbit Alexa FluorTM 488, Thermofisher, cat no., A-11034) expression according to TNF-α stimulation. (e) (d) is a graph showing the green fluorescence intensity. (f) Representative fluorescence images showing dHL-60 cell transendothelial migration in control and inhibitor (100 nM A-205804, Selleckchem, cat no, S2885; 10 μM KF 38789, TOCRIS, cat no, 2748)-treated groups. A-205804 selectively inhibits the surface expression of cell adhesion molecules ICAM-1 and E-selectin on human endothelial cells. KF 38789 selectively inhibits the surface expression of cell adhesion molecule P-selectin on human endothelial cells. (g) Quantification of the number of migrated dHL-60 cells in the control and inhibitor-treated groups is shown (** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

[0040] Figure 16 shows dHL-60 cell transendothelial migration without TNF-α treatment. (a) Representative fluorescence images show dHL-60 cell transendothelial migration in the NT and poly(I:C) groups. (b) Quantification of the number of migrated dHL-60 cells in the NT and poly(I:C) groups.

[0041] Figure 17 shows the results of the analysis of migration patterns according to distance. (a) The migration distance ranges were set to 0-50 μm, 50-200 μm, and ≥200 μm, and were measured by displacement analysis for cells treated with a single chemoattractant. (b) The results of the displacement analysis for cells treated with a combination of chemoattractants (IL-8, fMLP, C5a, and LTB4) are shown (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

[0042] Figure 18 shows the results of a comparative assay of dHL-60 cell transendothelial migration using plasma from patients with chronic obstructive pulmonary disease (COPD). (a) Quantification of cytokines and chemokines produced in the plasma of healthy subjects and COPD patients using ELISA. (b) Experimental setup for comparing dHL-60 cell transendothelial migration induced by healthy or COPD plasma. (c) Representative confocal microscopy images showing dHL-60 cell transendothelial migration between healthy subjects and COPD patients. (d) Number of migrated dHL-60 cells and displacement analysis in healthy and COPD patients. (e) Representative images of dHL-60 cell migration in serially diluted plasma from healthy subjects and COPD patient 1. (f) Quantification of the number of migrated dHL-60 cells in serially diluted plasma from healthy subjects and COPD patient 1. (g) Experimental setup used to analyze the drug efficacy of MK-7123 on dHL-60 cell migration. (h) Representative confocal microscopy images of dHL-60 cell transendothelial migration in the control and MK-7123 treated groups. (i) The number of migrated dHL-60 cells in the control and MK-7123 treated groups is shown (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

[0043] Hereinafter, preferred embodiments of the present invention will be described in detail. Furthermore, the following description includes numerous specific details, such as specific components, but these are provided to facilitate a more comprehensive understanding of the present invention. It will be apparent to those skilled in the art that the present invention can be practiced without these specific details. Furthermore, in describing the present invention, detailed descriptions of known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the present invention.

[0044]

[0045] FIG. 1 shows a perspective view (100) of a microfluidic device according to one embodiment of the present invention, and FIG. 2 shows a layout diagram of a gel channel, a common channel, and a comparison channel provided inside a microfluidic device according to one embodiment of the present invention.

[0046] As illustrated in FIGS. 1 and 2, a preferred embodiment of a microfluidic device (100) for investigating cell movement according to the present invention provides a microfluidic device characterized in that a gel channel (Gel channel, 150) having a first height (H'2) is arranged in parallel with a common channel (Common channel, 120) having a second height (H1) on one side thereof, and a comparative channel (Comparative channel, 130) having a second height (H1) on another side thereof, and ports (110, 140) capable of accommodating a liquid while allowing the injection and discharge of a liquid are provided at both ends of the gel channel, the common channel, and the comparative channel, and the common channel (120) and the comparative channel (130) are distinguished by a substrate injected into the gel channel due to a height difference (H2) between the first height (H'2) and the second height (H1).

[0047] In one aspect, the present invention provides a microfluidic device capable of simultaneously testing an experimental group and a control group with two comparison channels.

[0048] In one aspect, the present invention provides a microfluidic device that forms a cavity structure between a gel channel and a common channel and a comparison channel, which is not separated by a fixed structure such as a phase guide, a pillar, a partition wall or a membrane.

[0049] In one aspect, the present invention can provide a microfluidic device in which a fixed structure such as a phase guide, pillar, partition or membrane is formed in a part or the entirety of each channel as needed between a gel channel and a common channel and a comparison channel.

[0050] The above port (110, 140) has a chamber having an inlet or an outlet, and the port (110, 140) capable of receiving liquid while allowing the injection and discharge of liquid can be filled until the solution is discharged when injected from one side.

[0051] As illustrated in Fig. 3, the gel channel is formed in a straight line and has ports (110) at both ends, and the ports (110) include a chamber (201) having an inlet (202) or an outlet (202). In addition, the common channel (120) and the comparison channel (130) have ports (140) at both ends, and have a media inlet (204) and a media chamber (203).

[0052] FIG. 4 is a side view showing the difference between the first height (H2) and the second height (H1) of the microfluidic device according to one embodiment of the present invention, in which the gel channel (150), the common channel (120), and the comparison channel (130) are arranged without a fixed structure.

[0053] As illustrated in FIG. 4, the present invention provides a microfluidic device in which the first height (H2) of the gel channel is higher than the second height (H1). Here, the height difference (H2) is preferably 1.5 to 3 times higher than the second height, and most preferably 1.7 to 2.8 times. In some embodiments, the second height (H1) is 60 to 250 μm, and the first height (H2) is 60 to 260 μm higher than the second height (H1). That is, the first height is in the range of 120 to 510 μm. Here, the height difference (H2) means the difference between the first height (H'2) and the second height (H1), that is, H'2 = H1 + H2. (See FIG. 9 d)

[0054] Additionally, the present invention provides a microfluidic device in which the width of the gel channel is equal to or wider than the widths of the common channel and the comparison channel. Preferably, the gel channel (D1) is 1.1 to 1.5 times wider than the common channel (D2) and the comparison channel (D2), and more preferably, 1.1 to 1.3 times wider than the common channel (D2).

[0055] In another aspect, the present invention provides a microfluidic device characterized in that the gel channel is filled with a matrix. The matrix is ​​preferably a hydrogel, and more preferably, the hydrogel is at least one selected from the group consisting of collagen, fibrin, alginate, gelatin, laminin, agarose, fibronectin, polyacrylamide, polyanhydride, hyaluronic acid, and polypeptide. By using the above matrix in the gel channel of the present invention, a straight gel channel is formed in the height and width of the gel channel, and each channel is formed distinctly within the cavity structure without invading the common channel and the comparative channel.

[0056] As shown in Fig. 6, an example of a process of injecting an ECM mixture into a microfluidic device is shown by pushing in one direction and pulling in the other direction, and as shown in Fig. 7, an example of a process of injecting an ECM mixture into a microfluidic device is shown by pushing in both directions, an ECM mixture was used to mimic a substrate, and specifically, a 2.0 mg / mL collagen-based hydrogel containing fibronectin (10 μg / mL) was prepared as follows: 96 μL of distilled water, 20 μL of 10X phosphate buffered saline (PBS, pH 7.4, containing phenol red), 2.0 μL of 0.5 M sodium hydroxide (Sigma-Aldrich), 80 μL of collagen type I (5 mg / mL, Ibidi, Grafelfing, Bayern, Germany), and 2 μL of fibronectin (1 mg / mL, Sigma-Aldrich). This hydrogel was slowly injected into the gel channel of the device and polymerized for 1 hour at 37°C, 5% CO2.

[0057] The chip injected with the above substrate is placed on a plate, and the plate is placed in a 37°C incubator and incubated for 1 hour to allow complete gelation.

[0058] In another embodiment, the present invention provides an organ-on-a-chip comprising the configuration of the microfluidic device.

[0059] In the present invention, the term "biomimetic organ-on-a-chip" refers to a known "microphysiological system" or a microfabricated platform designed to model the functional units of an organ in vitro. A biomimetic organ-on-a-chip enables intimate contact between different cell types, such as between epithelium and vascular endothelium, while simultaneously generating spatiotemporal gradients of chemical and mechanical strain to mimic organ function.

[0060] FIG. 8 shows a comparative inflammation-on-a-chip (IoC) model for measuring neutrophil transendothelial migration (NTEM), and FIG. 9 shows the fabrication and preparation of the comparative inflammation-on-a-chip. As such, the microfluidic device of the present invention includes comparative channels 1 and 2, so that experimental and control drugs can be injected simultaneously to compare the results of cell migration simultaneously, and culture medium is injected into the common channel, and medium with or without experimental drug is injected into the comparative channel, so that the effect of the drug can be confirmed through cell migration at two points (Area 1 and Area 3). As a specific example, FIG. 9g shows FITC-dextran diffusion analysis under conditions with and without an endothelial barrier, and the results can be confirmed in Area 1 to Area 3.

[0061] In another aspect, the present invention provides a method for measuring cell movement using a microfluidic device, comprising: 1) forming a matrix in a gel channel; 2) injecting vascular cells into one side selected from a common channel and a comparative channel to form a blood vessel wall; 3) injecting cells capable of movement into the side of 2) where the blood vessel wall has been formed; and 4) injecting a substance capable of inducing cell movement into the side where the blood vessel wall has not been formed; wherein the gel channel is capable of allowing diffusion movement of the substance injected into the common channel and the comparative channel.

[0062] The above barrier-forming cells include all cells capable of forming a barrier in animals including humans, and are preferably, but not limited to, vascular endothelial cells.

[0063] The term "channel" as used herein refers to a microfluidic channel, a passage formed within a chip. A channel has a volume defined by its width, height, and length, at least one of which is less than a millimeter in size.

[0064] Here, the term "channel" includes, in addition to linear channels, channels having one or more branched, extended portions, and curved channels. In the present invention, the "channel" includes a port through which a volume of a liquid-type injectant can be injected. The volume of the substrate injected into the microfluidic channel is typically in the microliter or sub-microliter range. In some embodiments, the cross-sectional dimension of the channel is less than 1 millimeter, less than 500 micrometers, less than 100 micrometers, less than 50 micrometers, or less than 25 micrometers.

[0065] The above blood vessel wall may be formed on at least one selected from the group consisting of a substrate surface, an upper inner side of the channel, an inner side of the channel, and an inner lower side of the channel.

[0066] The above-mentioned mobile cells include all types of cells capable of moving in a living body, and may preferably be at least one selected from the group consisting of neutrophils, monocytes, macrophages, and cancer cells. The cancer cells are not particularly limited in type and nature, but may include, for example, breast cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, and bone cancer.

[0067] The substance capable of inducing the above cell migration may be an inflammatory factor or a cancer metastasis inducing factor.

[0068] The above 'inflammatory factor or cancer metastasis inducing factor' has no particular limitations in type, nature, etc., but may include, for example, IL-8, complement component 5a (C5a), leukotriene B4 (LTB4), N-formylmethionine-leucyl-phenylalanine (fMLP) or a combination of the four substances.

[0069] The above comparison channel may be a group in which conditions selected from a group consisting of an untreated group, a group treated with a substance that induces cell migration, a group treated with a substance that inhibits cell migration, and a group treated simultaneously with a substance that induces and inhibits cell migration can be compared.

[0070]

[0071] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0072]

[0073] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0074]

[0075] <Example 1> Preparation of the 3D-printed mold

[0076] 3D printing molds were designed in Rhinoceros (Rhino 7, Robert McNeel and Associates, Seattle, WA, USA). Each mold was used for polydimethylsiloxane (PDMS) casting to manufacture slide-sized devices. A ProJet MJP 2500 Plus printer (3D Systems, Valencia, CA, USA) was used to produce industrial-grade transparent resin (VisiJet ® M2S-HT90, 3D Systems) and support material (VisiJet ®The mold was printed using a M2 SUP, 3D Systems. The print consists of a resin that forms the structure and a support wax material. The support material was removed by sonication in a 65°C bath containing EZ Rinse-C solution (3D Systems) for 5 minutes in a 70°C oven for 2 hours. The mold was washed with soap, ethanol, and distilled water and dried by N2 blowing. The mold was post-cured by heat treatment in an 80°C oven. A 365 nm UV irradiation system (Bio-Link BLX-365, Villber-Lourmat, Eberhardzell, Germany) was used at 90 J / cm 2 UV post-curing tests were performed in the mold, but were ineffective. The post-cured mold was treated with a plasma (COVANCE, FemtoScience, Hwaseong, Korea) at 100 W for 1 min with a 200 sccm O2 flow (process pressure: 1.2 Torr), and then 60 μL of trichloro(1H,1H,2H,2H-perfluorooctyl)silane (Sigma-Aldrich, St. Louis, MO, USA) was placed in a vacuum desiccator (Φ 229 mm, LK Lab Korea Co., Korea). The vacuum desiccator was evacuated and placed in an oven at 70°C for 2 h for deposition.

[0077]

[0078] <Example 2> Fabrication of the comparative IoC device

[0079] Before using the 3D-printed mold, polyimide tape was attached to the back of the mold to block the holes. The silicone elastomer base and curing agent (Sylgard® 184, Dow Corning, Midland, MI, USA) were mixed at a ratio of 10:1 (w / w). The mixture was placed in a vacuum desiccator for 30 minutes to remove air bubbles, and then poured into the upper and lower molds. The mixture was then placed in a vacuum desiccator for another hour to remove air bubbles. The upper and lower molds were assembled and fully bonded using tweezers. The assembly was cured in a 70°C oven for 2 hours. After curing, the polyimide tape was removed, and the PDMS was separated from the 3D-printed mold. During the separation process, the PDMS could be easily removed by pushing it through the holes in the lower mold. After removing the unnecessary portion, a 3D PDMS structure with both upper and lower structures was obtained. Disassembling the mold under heated conditions is advantageous because it prevents the mold from bending and breaking due to heat. A detailed schematic of the manufacturing process is shown in Figure 5. The mold can be recycled and reused repeatedly without post-curing or coating. The PDMS product was washed, treated with oxygen plasma (100 W, 200 sccm oxygen flow for 1 minute), and then assembled onto a glass slide. The chip was vacuum-packed and stored at room temperature.

[0080]

[0081] <Example 3> Preparation of the extracellular matrix on a chip

[0082] The chips were sterilized and surface hydrophobicity restored by incubating them in an oven at 180°C for 2 h, and then cooled to room temperature before use in experiments. To mimic the ECM, a 2.0 mg / mL collagen-based hydrogel containing 10 μg / mL fibronectin was prepared as follows: 96 μL of distilled water, 20 μL of 10X phosphate-buffered saline (PBS, pH 7.4, containing phenol red), 2.0 μL of 0.5 M sodium hydroxide (Sigma-Aldrich), 80 μL of collagen type I (5 mg / mL, Ibidi, Grafelfing, Bayern, Germany), and 2 μL of fibronectin (1 mg / mL, Sigma-Aldrich). This hydrogel was slowly injected into the gel channel of the device and polymerized for 1 h at 37°C in 5% CO2. To prevent contamination, the chips were used in square dishes. The protocol for reproducible ECM loading is as follows.

[0083]

[0084] - Protocol: ECM Loading Guide

[0085] 1. Preparation of ECM mixture

[0086] The composition of the ECM mixture used in the present invention is as shown in Table 1.

[0087]

[0088] Volume (㎕) Maker Deionized water96.0Sigma-Aldrich10

[0089]

[0090] The present invention tested collagen concentrations ranging from 1 mg / mL to 4 mg / mL, all of which were limited and performed well. A collagen concentration of 2 mg / mL was ultimately selected and used, as this condition demonstrated proper migration. The composition of the hydrogel can be adjusted depending on the intended use.

[0091] *Note: The volume added will vary depending on the pipetting style (tip immersion depth or tip surface coating), so adjust the volume of 0.5 M NaOH solution appropriately while observing the color of the ECM mixture. Mix until the entire solution is uniform in color; the final ECM mixture should be a faint pink color.

[0092] *Caution: All components should be kept cold on ice or an ice shelf. Be careful not to create bubbles when mixing. The ECM mixture should be used within 30 minutes to ensure reproducible gelation.

[0093]

[0094] 2. Injecting ECM mixture into the chip

[0095] Both methods can be used for ECM mixture loading.

[0096] *Caution: Excessive injection or suction force may cause ECM mixture to leak into the media channel. Work slowly; the injection or suction step in this experiment takes approximately 10 seconds.

[0097]

[0098] 2-1. Push in one direction, pull in the other direction (see Figure 6)

[0099] Using a 10 μL pipette, dispense 10 μL of ECM and insert the pipette tip into the hydrogenation well. Once the top surface is reached, connect the top portion of the ECM until it exceeds the mobile area that contacts the media channel. Keep the pipette tip short and insert the remaining ECM into the front.

[0100] Then, insert the pipette tip into the opposite gel inlet until it reaches the bottom of the chamber. Slowly aspirate the ECM mixture to fill the remaining gel channel, and continue to aspirate some of the gel into the tip. The mold is designed to fit the pipette tip to facilitate smooth aspiration of the ECM mixture. Finally, lift the pipette tip and inject the ECM mixture back into the gel inlet and chamber. Placing the chip horizontally ensures that the level is the same.

[0101] *Caution: If the ECM mixture does not flow in, check that the pipette is not fully inserted or that the front of the ECM does not exceed the movement area. If the ECM mixture does not exceed the movement area, the pipette will apply negative pressure to the empty space in the ECM channel.

[0102]

[0103] 2-2. Push in both directions (see Figure 7)

[0104] Using a 10 μL pipette, aspirate 5 μL of ECM mixture and insert the pipette tip into one side of the gel inlet until it touches the chamber bottom. Slowly inject some of the ECM mixture until it reaches the center of the channel, then slightly lift the pipette tip and inject the remaining ECM mixture into the gel inlet and chamber.

[0105] Aspirate another 5 μL of the ECM mixture and insert the pipette tip into the other side of the gel inlet until it reaches the bottom of the chamber. Slowly inject some of the ECM mixture until the two gel fronts merge, then slightly lift the pipette tip and inject the remaining ECM mixture into the gel inlet and chamber.

[0106] Method 2-2 is a more common protocol for existing chips, but we preferred Method 2-1 because air bubbles occasionally become trapped where the two ECM wires merge in Method 2-2 (at about one frequency per 30 tests). This is because the ECM channel is higher than the two side channels, so air bubbles become trapped at the ceiling.

[0107]

[0108] 3. Gelation

[0109] To reduce evaporation, distilled water is added to the DW chamber. To prevent contamination during transport, the chip is placed in a dish, then placed in a 37°C incubator and incubated for 1 hour to allow complete gelation.

[0110] *Caution: To maintain the integrity of the ECM, care must be taken to avoid excessive shock during movement.

[0111]

[0112] <Example 4> Diffusion and permeability assay

[0113] 40 μL of Iscove's modified Dulbecco's medium (IMDM, ATCC) was injected into the common channel, and 60 μL of medium with or without FITC-dextran (10 μM, molecular weight: 10,000, Sigma-Aldrich) solution was injected into comparison channel 2 and comparison channel 1, respectively. Fluorescence intensity was monitored at three representative locations at 30-min intervals for 5 h.

[0114]

[0115] <Example 5> Cell culture

[0116] HL-60 cells (CCL-240, ATCC, Manassas, VA, USA), a representative phenotype suitable for neutrophil migration, were cultured at 1 x 10 6 75 cm at a density of cells / mL 2Cells were seeded into flasks. Cells were cultured in IMDM containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% (v / v) penicillin / streptomycin (Sigma-Aldrich) at 37°C in 5% CO2. HL-60 cells were differentiated into neutrophil-like cells by adding DMSO to a final concentration of 1.3% and cultured for 7 days. Other differentiation conditions (10 μM ATRA and 500 μM dbcAMP) were also tested. Human umbilical vein endothelial cells (HUVECs; Lonza, Basel, Switzerland), which have a phenotype suitable for studying neutrophil endothelial transmigration, were cultured in EBMTM-plus growth medium containing BulletKitTM (Lonza). Human bronchial epithelial cells (Calu-3, ATCC), a phenotype suitable for studying lung inflammation, were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% (v / v) penicillin / streptomycin, 1X minimal essential medium (MIM) non-essential amino acids (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 1 mM sodium pyruvate (Gibco). HL-60 cells were used from passage 6 to 15. HUVEC and Calu-3 cells were used from passage 3 to 10.

[0117]

[0118] <Example 6> Neutrophil migration experiments

[0119] Differentiated HL-60 (dHL-60) cells were suspended and dead cells were removed using a dead cell removal kit (Miltenyi Biotec, North Rhine-Westphalia, Germany) according to the manufacturer's instructions. dHL-60 cells (2 X 10 660 μL at a density of cells / mL was injected into the channel. To study chemoattractant-induced neutrophil migration, 40 μL of medium with or without chemoattractants was added to the comparison channels. Comparison channel 1 was filled with control medium, and comparison channel 2 was filled with medium containing 50 ng / mL IL-8 (PeproTech, Cranbury, NJ, USA) and 10 nM complement component 5a (C5a, R&D Systems, Minneapolis, MN, USA), 10 nM leukotriene B4 (LTB4, Cayman Chemical, Ann Arbor, MI, USA), 1 μM N-formylmethionine-leucyl-phenylalanine (fMLP, Sigma-Aldrich), or a combination of the four chemoattractants. To study chemoattractant receptor blockade, dHL-60 cells were pre-incubated with CXCR2 antibody (1 μg / mL, R&D Systems), Boc-MLP (100 μM, Sigma-Aldrich), DF2593A (1 μM, Sigma-Aldrich), or LY223982 (10 μM, Cayman Chemical) for 1 h at 37°C in 5% CO2 and then applied to the control channel. Medium (40 μL) containing chemoattractants was injected into the common channel. The device was placed in a CO2 incubator for 2 h to allow cell migration into the ECM. For endothelial barrier formation, HUVECs were detached using 0.25% trypsin / EDTA solution and seeded at 5 × 10 6After injecting 20 μL of a cell / mL density into the microchannel of the device, the device was immediately tilted for 2 h to promote endothelial barrier formation on the gel side. The device was then laid flat and cultured for 24 h to allow monolayer formation. After endothelial barrier formation, NTEM was observed in the presence of 1 μg / mL polyinosinic:polycytidylic acid (poly(I:C); InvivoGen, San Diego, CA, USA) conditioned medium and plasma from COPD patients. dHL-60 cells were pre-incubated with CXCR2 antagonists for 1 h at 37°C, 5% CO2, then added to the control channel, and the device was placed in a CO2 incubator for 4 h to perform NTEM analysis.

[0120]

[0121] <Example 7> Flow cytometry

[0122] HL-60 cells and HUVECs were stained with Fixable Viability Dye eflour 780 (1:1000, Thermo Fisher Scientific) at 4°C for 30 min. Cells were then incubated with fluorescently conjugated primary antibodies anti-CXCR1-APC (Cat. No. 320612), anti-CXCR2-PE-Cy7 (Cat. No. 320716), anti-CD11b-BV605 (Cat. No. 301332), anti-C5aR-PerCP-Cy5 (Cat. No. 344312), anti-ICAM-1 (Cat. No. 353108), anti-CD62E (Cat. No. 336012), anti-CD62P (Cat. No. 304920) (BioLegend, San Diego, CA, USA), anti-LTB4R-FITC (Cat. No. FAB099F), anti-FPR1-APC (Cat. No. FAB3744A), or anti-FPR2-PE (Cat. No. FAB3744A) (R&D Systems) were stained for 30 min at 4°C in FACS buffer containing 2% (v / v) BSA and 2 mM EDTA in DPBS. The suspended single cells were washed twice with FACS buffer, and then the cells were detected with a BD LSRFortessa™ cell analyzer (BD Biosciences, Franklin Lakes, NJ, USA). FlowJo TM Data were analyzed using v10 software (BD Biosciences).

[0123]

[0124] <Example 8> Cell staining and microscope imaging

[0125] dHL-60 cells were stained with Calcein-AM (2 μM, Thermo Fisher Scientific). The endothelial barrier formed by HUVECs was fixed in 10% neutral buffered formalin (Sigma-Aldrich), and then the cells were blocked in 0.1% Triton X-100 (PBS) containing 4',6-diamidino-2-phenylindole (DAPI, 100 ng / ml, Sigma-Aldrich) and rhodamine phalloidin (Thermo Fisher Scientific). TM Staining with X-100 for 30 min was performed. The migration of dHL-60 cells was monitored using a fluorescence microscope (Olympus IX-73, Tokyo, Japan). Images were analyzed (number of migrated cells and displacement) using MetaMorph software version 7.10.5.476. 3D and maximum projection images were analyzed using a confocal laser scanning fluorescence microscope (Stellaris 8, Leica Microsystems, Wetzlar, Germany).

[0126]

[0127] <Example 9> Enzyme-linked immunosorbent assay (ELISA)

[0128] Calu-3 cells (1 X 10 5Cells / mL) were seeded in 6-well plates and cultured for 4 days to reach approximately 90% confluency. Calu-3 cells were stimulated with or without poly(I:C) for 24 h. Supernatants were harvested at the end of stimulation. COPD plasma samples (Cat. No. 991-58-S-COPD) were purchased from Lee BioSolutions, Maryland Heights, MO, USA. Details of patient characteristics are shown in Table 2. TNF-α, IL-6, IL-1β, and IL-8 concentrations were measured in Calu-3 cell culture supernatants with or without poly(I:C) stimulation or in serum from COPD patients using ELISA kits (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0129]

[0130] Cat. NumberMatrixSample IDAgeGenderRace991-58-S-COPDPlasma183FemaleCaucasian276MaleCaucasian388MaleCaucasian477FemaleCaucasian591MaleCaucasian

[0131]

[0132] <Example 10> Statistical analysis

[0133] Statistical analysis was performed using Prism 9 (GraphPad Software, San Diego, CA, USA). Statistical analyses were performed using one-way ANOVA or Student's t-test. All results are expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0134]

[0135] <Experimental Example 1> Design and preparation of a comparative inflammation-on-a-chip using 3D printing

[0136] To compare NTEM in COPD with NTEM under healthy conditions in the same device, a novel IoC was designed (see Fig. 8a). While NTEM is absent in healthy subjects, NTEM is induced in COPD patients by inflammatory chemoattractants between the extracellular matrix (ECM) and blood vessels. The IoC of the present invention comprises a channel that mimics the physiological 3D microenvironment of COPD-induced neutrophil migration across the endothelial barrier. This device incorporates a COPD 3D microenvironment and utilizes neutrophil-like cells expressing a normal surface receptor found on endogenous neutrophils to monitor NTEM (see Fig. 8b).

[0137] Previous OoC devices for individual analysis were developed with fixed structures, such as pillars or phase guides. Pillar structures can interfere with the formation of a continuous, intact endothelium. Phase guide structures are not optimal for identifying and monitoring neutrophil migration induced by inflammation, as they can interfere with cell migration. Furthermore, reproducibility is a critical issue in comparative analyses of pharmacological effects using OoCs. Therefore, a complete 3D interface between the artificial compartments of the IoC is necessary for reproducible and comparative analyses of neutrophil migration.

[0138] 3D printing technology enables the precise formation of microstructures made of various materials and allows for customization of designs; it allows for rapid modifications; it means a short manufacturing process, low cost, and minimal waste. For these reasons, a 3D-printed mold was used to prepare the IoC (see Figure 9). The IoC was developed to mimic the microenvironment, as shown in Figure 8, by including a common gel and two comparison channels. A single module of the IoC is shown on the left side of Figure 9a. The gel channel (blue) in the extracellular region was positioned in the middle of the IoC. The common channel (red) was positioned on one side. The two comparison channels (yellow and green) were positioned on the other side of the common channel of the IoC (right side of Figure 9a). The comparison channel and the common channel were equally adjacent to the gel channel. The cross-sectional structure of the media chamber and inlet of this IoC was designed to facilitate the smooth injection of biomaterials using a micropipette (see Figure 9b). The phaseguide and pillar structures in the migratory region impede the diffusion of chemoattractants and cell migration. This feature makes the IoC model of the present invention the first to mimic COPD-induced NTEM in a 3D environment with a complete 3D interface between the endothelial barrier and the ECM.

[0139] The 3D printed mold for forming each channel in the moving region is shown in Figure 9c. The height of each channel was determined by examining the success rate of ECM confinement in the IoC. The heights of the common and comparison channels (H1) were maintained at 96–224 μm, and the stacking height above H1 (H2) was maintained at 64–256 μm (see Figure 9d). Heights of H1 and H2 were selected for the IoC. A representative image of ECM confinement is shown in Figure 9e. Four identical modules were integrated into a single IoC to enable replication experiments. The 3D printed mold consists of two parts. The upper part contains a guide structure for PDMS cutting and channels for the gel and media, while the lower part contains a chamber with an inlet structure for the gel and media (see Figure 10). The optimal conditions for post-processing the 3D printed mold were determined to be 80°C for 24 h (see Figure 11). The channel images of the IoC are shown in Fig. 9f. To validate the comparative analysis setup, the diffusion of the injected solutions, including IMDM medium and FITC-dextran solution, was analyzed by measuring the fluorescence intensity (see Fig. 9g). The fluorescence signal was observed only in region 1 (see left in Fig. 9g) and gradually increased over 1 h before saturation, whereas a weak signal was observed in region 2 after 4 h without an endothelial barrier. In contrast, in the presence of an endothelial barrier composed of HUVEC cells in comparison channels 1 and 2 of the IoC (see right in Fig. 9g), a weak signal was detected only in region 1. These results demonstrated that the endothelial barrier in each comparison channel inhibited the diffusion of the FITC-dextran solution, indicating that the diffusion characteristics of the IoC are suitable for performing comparative analysis with NTEM.

[0140]

[0141] <Experimental Example 2> Expression of representative surface markers on differentiated HL-60 cells

[0142] Primary neutrophils from healthy human subjects have been used in previous studies to analyze neutrophil migration characteristics. Because primary neutrophils have a short lifespan and cannot be cryopreserved or expanded, they must be freshly isolated from peripheral blood. Furthermore, isolated primary cells produce variable experimental results in a donor-dependent manner. Therefore, in the present invention, we chose to use an appropriate cell line and determined that it expresses the appropriate chemoattractant receptor.

[0143] HL-60 cells, derived from acute promyelocytic leukemia, are the most common cell line used in neutrophil research. They can be differentiated into mature granulocytic lineages using agents such as all-trans retinoic acid (ATRA), dibutyryl cyclic adenosine monophosphate (dbcAMP), or dimethyl sulfoxide (DMSO). Previous studies on HL-60 cell differentiation have not clearly addressed both the expression of chemoattractant receptors and the viability of differentiated cells. Therefore, in the present invention, HL-60 cells were treated with the differentiation inducers ATRA, dbcAMP, and DMSO for 7 days, and receptor expression patterns and viability were examined (see Figure 12). The basic gating strategy for flow cytometry is shown in Figure 12a. The viability of dHL-60 cells was further analyzed under differentiation conditions using ATRA, dbcAMP, and DMSO (see Figure 12b). The viability rates were as follows: undifferentiated HL-60 cells, 89%; dHL-60 cells treated with 10 μM ATRA, 37%; dHL-60 cells treated with 500 μM dbcAMP, 33%; dHL-60 cells treated with 1.3% DMSO, 70%. HL-60 cells treated with DMSO showed higher survival rates than cells treated with ATRA or dbcAMP.

[0144] The expression of CD11b, a surface marker of differentiated neutrophil-like cells, was analyzed in HL-60 cells treated with DMSO, dbcAMP, and ATRA (Fig. 12c,d). CD11b expression was significantly increased by DMSO or ATRA treatment, but not by dbcAMP treatment. Furthermore, the expression patterns of representative chemotactic receptors (C5aR, LTB4R, CXCR1, CXCR2, FPR1, and FPR2) were examined to determine whether dHL-60 cells treated with each inducer represented neutrophil-like cells. dHL-60 cells were stained with the appropriate fluorophore-labeled primary antibodies. ATRA and dbcAMP treatment increased the expression of some, but not all, receptor types. In contrast, DMSO consistently increased the expression of representative chemoattractant receptors (C5aR, LTB4R, CXCR1, CXCR2, FPR1, and FPR2) in dHL-60 cells. In summary, these results demonstrate that DMSO treatment of HL-60 cells maintains viability and induces a chemoattractant receptor expression pattern similar to that of mature neutrophils. The in vitro conditions of the present invention can be well-established for the study of inflammatory diseases, as they appropriately express chemoattractant receptors responsible for neutrophil migration.

[0145]

[0146] <Experimental Example 3> Neutrophil migration in the IoC model

[0147] To evaluate neutrophil migration induced by chemoattractants in the IoC model of the present invention, we investigated neutrophil migration induced by a gradient of chemoattractants (see Fig. 13a). The gel channels were filled with collagen to form an ECM. dHL-60 cells were injected into the common channel, while medium and chemoattractants (IL-8, fMLP, C5a, and LTB4) were added to control channels 1 and 2, respectively. dHL-60 cell migration was measured using a fluorescence microscope for 2 hours (see Figs. 13b,c). Treatment of cells with IL-8, fMLP, C5a, or LTB4 alone or in combination significantly induced neutrophil migration compared to the control medium. To evaluate neutrophil migration induced by the interaction between chemoattractants and their cognate receptors, the effect of a chemoattractant receptor blocker (CRB) was evaluated (see Fig. 13d). CXCR2 antibodies, Boc-MLF, DF293A, and LY22398 were used as antagonists for IL-8, fMLP, C5a, and LTB4, respectively. Each antagonist inhibited chemoattractant-induced neutrophil migration (see Figures 13e and f). These results demonstrated that neutrophil migration and its inhibition can be demonstrated in an IoC model without an endothelial barrier. Furthermore, these results imply that the in vitro conditions used to differentiate HL-60 cells resulted in the appropriate expression of chemoattractant receptors mediating neutrophil migration.

[0148]

[0149] <Experimental Example 4> Blockade effect of CXCR2 inhibitors underin vitroinflammatory conditions induced by poly(I:C)

[0150] Next, NTEM analysis was performed using IoCs with an endothelial barrier under in vitro inflammatory conditions (see Figure 14). HUVECs constituting the endothelial layer expressed CD31, an endothelial cell marker (Figure 15a). TNF-α induces high levels of expression of adhesion proteins such as ICAM-1, VCAM-1, and E-selectin, thereby inducing leukocyte adhesion to endothelial cells. The expression levels of the adhesion proteins ICAM-1, E-selectin (CD62E), and P-selectin (CD62P) were also investigated in a 2D in vitro culture system in response to TNF-α treatment. ICAM-1 and CD62E levels increased significantly and progressively with the addition of TNF-α. However, CD62P was basally highly expressed in endothelial cells (Figures 15b and c).

[0151] Previous studies on physiological neutrophil extravasation under inflammatory conditions have used poly(I:C), a Toll-like receptor 3 (TLR3) agonist that induces cellular secretion of proinflammatory mediators such as cytokines and chemoattractants. In vitro culture conditions representing lung inflammation were established using poly(I:C) and Calu-3 cells (Fig. 14a). The expression levels of TNF-α, IL-1β, IL-6, and IL-8 in conditioned medium of Calu-3 cells treated with 1 μg / mL poly(I:C) for 24 h were measured by ELISA (Fig. 14b). Poly(I:C) significantly increased the expression of IL-6 (4.16-fold) and IL-8 (2.40-fold), but not TNF-α and IL-1β, compared to untreated control medium.

[0152] To evaluate how in vitro inflammatory conditions induce NTEM, we prepared IoCs containing an endothelial barrier formed by HUVEC cells cultured in a common channel. Control and conditioned media were then injected into Comparison Channel 1 and Comparison Channel 2, respectively (Fig. 14c). The HUVEC cell layers on the upper, lower, and lateral sides of the common channel were observed as a 3D structure using a confocal microscope (upper image of Fig. 14d). These results demonstrated that HUVEC cells cultured in the common channel formed a barrier that mimicked the 3D structure of the vascular lumen. Next, we investigated the effects of NTEM and TNF-α on poly(I:C)-induced inflammation. TNF-α-stimulated endothelial monolayers generally showed higher expression of ICAM-1 in the IoC model than in the control model (Fig. 15d and e). Furthermore, the number of migrated dHL-60 cells was lower in the adhesion protein (ICAM-1, CD62E, and CD62P) inhibitor-treated group than in the untreated control group (Fig. 15f and g). As expected, in the absence of TNF-α, dHL-60 cells did not significantly migrate in the control or poly(I:C)-conditioned medium (Fig. 16). Therefore, TNF-α stimulation was essentially required for dHL-60 cell migration across the endothelial barrier in the poly(I:C)-induced in vitro inflammation model.

[0153] Next, we measured the distance dHL-60 cells migrated across the endothelial barrier by measuring the difference between the initial and final positions of migrated cells (Fig. 14d,e). The CXCR2 / IL-8 axis is involved in the initial neutrophil chemotaxis from healthy tissues to sites of inflammation and is dependent on chemokine gradients in vascular tissues, whereas the formyl peptide (FPR1 / fMLP axis) governs the final migration step toward inflamed tissues. The distance ranges were set as 0–50 μm (near the endothelium), 50–200 μm, and >200 μm (migration toward sites of inflammation) (Fig. 14c). The number of migrated cells was significantly higher at each displacement in poly(I:C)-containing medium than in the control medium. This result was similar to the displacement induced by Combi treatment (Fig. 17). Therefore, the IoC model of the present invention, which includes the endothelial barrier and ECM, can be used to study NTEM mimicking lung inflammation, and is the first OoC to utilize such in vitro inflammatory conditions for NTEM studies.

[0154] IL-8 is a chemokine highly expressed in the blood, bronchoalveolar lavage fluid, and sputum of COPD patients and induces neutrophil infiltration into inflamed lung tissue. Inhibitors of CXCR2, the IL-8 receptor, effectively block neutrophil infiltration in mouse models and human clinical trials. MK-7123 and AZD5069, used as CXCR2 antagonists, have been tested as candidate mechanisms for COPD drug development. To investigate the pharmacological effects of CXCR2 antagonists on neutrophil migration, dHL-60 cells were treated with antagonists or controls for 2 hours, and then injected with TNF-α into Comparator Channel 2 (antagonist) and Comparator Channel 1 (control), which contain the endothelial barrier, respectively (Fig. 14f). NTEM induced by poly(I:C)-conditioned medium was examined in the presence of CXCR2 inhibitors. MK-7123 significantly inhibited NTEM (p-value = 0.013, Fig. 14g), whereas AZD5069 tended to inhibit NTEM (p-value = 0.1), and SB225002 had no effect (p-value = 0.3) (Fig. 14h). Therefore, MK-7123 was used in a study of plasma samples from COPD patients.

[0155]

[0156] <Experimental Example 5> NTEM responses in COPD patient samples and MK-7123 treatment

[0157] COPD-induced proinflammatory mediators promote spontaneous neutrophil infiltration into the lungs and subsequent alveolar destruction, contributing to the progressive deterioration of lung function. The degree of neutrophil infiltration in the lungs is correlated with the severity of COPD. Therefore, we measured the expression levels of proinflammatory mediators, such as TNF-α, IL-1β, IL-6, and IL-8, in the plasma of healthy subjects and COPD patients (n = 5) purchased from Lee BioSolutions (Figure 18a). There was significant patient-specific variability in the data; IL-6 and IL-8 expression levels were significantly increased in patients 1 (P1), P2, and P4, but not in P3 and P5. The increases in IL-6 and IL-8 levels in some patients were similar to the cytokine profile of poly(I:C)-induced in vitro inflammatory conditions (Figure 14b).

[0158] To evaluate NTEM in plasma from COPD patients in the IoC, dHL-60 cells and TNF-α were injected into the common channel after the endothelial barrier was formed (Fig. 18b). Plasma samples from healthy subjects were applied to comparison channel 1, and plasma samples from COPD patients were applied to comparison channel 2. NTEM was significantly higher in all plasma samples from COPD patients than in the samples from healthy subjects (Fig. 18c, d). Next, cell displacement was measured (right graph in Fig. 18d). This showed that although IL-6 and IL-8 expression levels did not increase in the P3 and P5 samples, dHL-60 cell displacement was higher in the patient samples. This result is consistent with the results of measuring dHL-60 cell migration distance under Combi treatment or poly(I:C) in vitro inflammatory conditions (Fig. 14e and Fig. 17). Results from patient samples suggest that the long migration distance (>200 μm) of dHL-60 cells in IoC may be due to the synergistic effects of known (IL-6 and IL-8) and unknown mediators. Use of a single chemoattractant did not result in significant cell displacement (Fig. 17).

[0159] Next, to determine the sensitivity of COPD-induced NTEM, plasma was serially diluted from P1 (Fig. 18e). NTEM occurred in the presence of diluted plasma (Fig. 18f), demonstrating that the IoC model of the present invention is sensitive even at low plasma concentrations. The pharmacological effect of MK-7123 on COPD-induced NTEM was then examined (Fig. 18g). MK-7123 treatment significantly inhibited COPD-induced NTEM, but not untreated control NTEM (Fig. 18h,i). Displacement was then measured (right graph of Fig. 18i), demonstrating that MK-7123 inhibited COPD-induced NTEM across all displacement ranges (0–50 μm, 50–200 μm, and >200 μm). MK-7123 inhibited the long-range migration observed in the present invention. These results suggest that IL-8 and / or CXCR2 play important roles in COPD-mediated NTEM and may be potential drug targets.

[0160]

[0161] <Experimental Example 6> Discussion

[0162] Previous cell migration measurement devices aimed at mimicking physiological conditions in vivo were developed using high-resolution (micrometer) photolithography methods. However, photolithography is suitable for single-layer structures. New photomasks not only require slight modifications to the structure, but also make it difficult to form complex structures, especially those taller than adjacent structural elements, using photolithography. Meanwhile, 3D printing technology can easily produce molds that create complex structures, such as those used for the upper and lower structures of the IoC fabricated in the present invention. The 3D printing method enabled the IoC to easily have gel channels with a height H2 greater than the height H1 (Figure 9c).

[0163] Many 3D cell migration assays have been developed that incorporate micropillar structures for gel confinement. In these devices, cell migration can occur only within the very narrow gaps between the micropillar structures, resulting in a low cell-to-gel channel interface ratio. An assay that incorporates a phase-guided structure to confine the gel has been developed, improving the cell-to-gel channel interface ratio to over 90%. Importantly, the H1 and H2 structures, which lack phase-guided and pillar structures, are employed in the IoC, enabling a complete 3D interface between the microchannels. The H1 and H2 structures also enable the formation of 3D structures, such as the vascular lumen. Furthermore, variability and reproducibility between in vitro and in vivo experiments are crucial for drug development and for determining the mechanisms of chemotaxis. The IoC of the present invention, which allows simultaneous comparison of the effects of two stimuli in a single device, may be more powerful than single experiments using previous OoCs. Therefore, most OoC devices used for comparative cell migration assays may require the H1 and H2 structures to more conveniently and fully implement a 3D microenvironment in a single device. The IoC model of the present invention also features a robust modular design, enabling real-time quantitative assessment of NTEM across a wide range of gel barriers and convenient manipulation for pharmacological applications.

[0164] Primary neutrophils isolated from healthy human subjects have limitations, such as a short lifespan and poor cryopreservation. Furthermore, results obtained using primary neutrophils may not be reproducible and may vary depending on the donor. To overcome these issues, we used HL-60 cells, a cell line isolated from patients with acute promyelocytic leukemia. These cells can be differentiated into mature granulocytes, resembling neutrophils, after treatment with ATRA, dbcAMP, or DMSO. Here, the present invention demonstrated that the expression of representative chemoattractant receptors, such as CXCR1, CXCR2, C5aR, LTB4R, FPR1, FPR2, and CD11b (Mac-1), in HL-60 cells is involved in neutrophil migration (Figure 12). Neutrophil migration was induced by the interaction between chemoattractants and their receptors. Treatment of dHL-60 cells with chemoattractants, such as IL-8, fMLP, C5a, and LTB4, either alone or in combination, significantly increased dHL-60 cell migration compared to untreated controls (Fig. 13). The results presented herein evaluated the characteristics associated with differentiation and migration of neutrophil-like cells.

[0165] Pulmonary inflammation is mediated by complex tissue-tissue interactions between the pulmonary airway epithelium and the vascular endothelium. This inflammatory response modulates the immune response to respiratory pathogens, leading to increased neutrophil infiltration and clinical exacerbation of lung disease. The complex interactions underlying lung inflammation have made it difficult to model the complex inflammation of human lung diseases such as asthma and COPD. The IoC model of the present invention can mimic human lung inflammation accompanied by an immune response associated with NTEM. Calu-3 cells, a human bronchial epithelial cell line, express TLR3, which recognizes double-stranded RNA (dsRNA). In particular, poly(I:C), a synthetic form of double-stranded RNA, stimulates TLR3, which subsequently activates NF-κB and produces cytokines and chemokines. Viral infections are a major cause of acute exacerbations of COPD. For example, rhinovirus is a major cause of the common cold and exacerbates the pathogenesis of asthma and COPD. Therefore, poly(I:C)-induced inflammation is an important in vitro model for studying lung inflammatory diseases, including COPD.

[0166] The pathogenic mechanisms of COPD involve a pulmonary inflammatory response mediated by gene expression and environmental factors, which leads to parenchymal damage and loss of elastic recoil. Among these mechanisms, spontaneous neutrophil infiltration into the lungs is implicated in the pathogenesis of COPD, and the level of infiltration correlates with COPD severity. Novel molecularly targeted therapies aimed at alleviating and treating COPD progression are under development. Clinical trials have been conducted to determine whether CXCR2 antagonist therapy is effective in COPD. MK-7123 and AZD5069 are two clinical trials designed to determine whether CXCR2-dependent neutrophil migration and NTEM inhibition are effective in treating COPD patients. In COPD patients, MK-7123 improved clinical symptoms, namely forced expiratory volume in 1 second (a measure of bronchodilator function). However, AZD5069 did not induce clinically relevant changes, and the effect of SB225002 in COPD patients has not been reported. The results obtained with these inhibitors in the IoC (Figures 14 and 18), where MK-7123 significantly inhibited NTEM and AZD5069 tended to inhibit NTEM, reflect the results of clinical studies of MK-7123 and AZD5069.

Claims

1. A gel channel (Gel channel, 150) having a first height (H'2) is arranged in parallel with a common channel (Common channel, 120) having a second height (H1) on one side thereof, and a comparative channel (Comparative channel, 130) having a second height (H1) is included on another side of the gel channel. At both ends of the above gel channel, common channel and comparison channel, ports (110, 140) capable of receiving liquid while allowing liquid injection and discharge are provided. A microfluidic device characterized in that the common channel (120) and the comparison channel (130) are distinguished by a substrate injected into the gel channel by a height difference (H2) between the first height (H'2) and the second height (H1).

2. In the first paragraph, the microfluidic device is characterized in that the comparison channel simultaneously tests the experimental group and the control group with two channels.

3. A microfluidic device according to claim 1 or 2, characterized in that a cavity structure is formed between the gel channel and the common channel and the comparison channel.

4. A microfluidic device according to claim 1 or 2, characterized in that the port (110, 140) has a chamber having an inlet or an outlet.

5. A microfluidic device according to claim 1 or 2, characterized in that the first height (H'2) is 1.7 to 2.8 times higher than the second height (H1).

6. A microfluidic device according to claim 1 or 2, characterized in that the width (D1) of the gel channel is equal to or wider than the width (D2) of the common channel and the comparison channel.

7. A microfluidic device according to claim 1 or 2, characterized in that the gel channel is capable of diffusion movement of a substance injected into the common channel and the comparison channel.

8. A microfluidic device according to claim 1 or 2, characterized in that the substrate is a hydrogel.

9. In the 8th paragraph, the hydrogel is characterized in that it is at least one selected from the group consisting of collagen, fibrin, alginate, gelatin, laminin, agarose, fibronectin, polyacrylamide, polyanhydride, hyaluronic acid, and polypeptide.

10. A gel channel (Gel channel, 150) having a first height (H'2) is arranged in parallel with a common channel (Common channel, 120) having a second height (H1) on one side thereof, and a comparative channel (Comparative channel, 130) having a second height (H1) is included on another side of the gel channel. At both ends of the above gel channel, common channel and comparison channel, ports (110, 140) for injecting and receiving liquid are provided. The common channel (120) and the comparison channel (130) are distinguished by the substrate injected into the gel channel due to the height difference between the first height (H'2) and the second height (H1). An organ-on-a-chip characterized in that the above gel channel enables diffusion movement of a substance injected into the common channel and the comparison channel.

11. In the 10th paragraph, the comparison channel is characterized by simultaneously testing the experimental group and the control group through two channels, an organ-on-a-chip.

12. An organ-on-a-chip according to claim 10 or 11, characterized in that a cavity structure is formed between the gel channel and the common channel and the comparison channel.

13. An organ-on-a-chip according to claim 10 or 11, characterized in that the port (110, 140) has a chamber having an inlet or an outlet.

14. An organ-on-a-chip according to claim 10 or 11, characterized in that the first height (H'2) is 1.7 to 2.8 times higher than the second height (H1).

15. An organ-on-a-chip according to claim 10 or 11, characterized in that the width (D1) of the gel channel is equal to or wider than the width (D2) of the common channel and the comparison channel.

16. An organ-on-a-chip according to claim 10 or 11, characterized in that the gel channel is capable of diffusion movement of a substance injected into the common channel and the comparison channel.

17. An organ-on-a-chip according to claim 10 or 11, characterized in that the substrate is a hydrogel.

18. An organ-on-a-chip according to claim 17, wherein the hydrogel is at least one selected from the group consisting of collagen, fibrin, alginate, gelatin, laminin, agarose, fibronectin, polyacrylamide, polyanhydride, hyaluronic acid, and polypeptide. 19.1) Step of forming a matrix in a gel channel; 2) A step of forming a blood vessel wall by injecting blood vessel cells into one side selected from among the common channel and the comparative channel; 3) a step of injecting movable cells into the side of the blood vessel wall formed in 2); and 4) a step of injecting a substance capable of inducing cell migration into the side where the blood vessel wall is not formed; A method for measuring cell movement using the microfluidic device of claim 1 or the biomimetic organ chip of claim 10, wherein the gel channel is capable of diffusion movement of a substance injected into a common channel and a comparison channel.

20. In the 19th paragraph, the cell movement measurement method characterized in that the comparison channel simultaneously tests the experimental group and the control group with two channels.

21. A cell migration measurement method according to claim 19, characterized in that the vascular cells are vascular endothelial cells.

22. A cell migration measurement method according to claim 19, characterized in that the blood vessel wall is formed on at least one selected from the group consisting of a substrate surface, an inner upper portion of the channel, an inner side of the channel, and an inner lower portion of the channel.

23. A cell migration measurement method according to claim 19, characterized in that the mobile cells are at least one selected from the group consisting of neutrophils, monocytes, macrophages, and cancer cells.

24. A method for measuring cell movement, characterized in that the substance capable of inducing cell movement in claim 19 is an inflammatory factor or a cancer metastasis-inducing factor.

25. In the 19th paragraph, the comparison channel is characterized in that conditions selected from a group consisting of an untreated group, a group treated with a substance that induces cell migration, a group treated with a substance that inhibits cell migration, and a group treated simultaneously with a substance that induces and inhibits cell migration can be compared. A cell migration measurement method.

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