Microfluidic system for simulating lung tissue

Abstract

This invention discloses a biomimetic system simulating lung tissue, its manufacturing method, and a method for controlling microfluidics using this biomimetic system. The biomimetic system comprises lung epithelial cells, lung fibroblasts, and human umbilical vein endothelial cells isolated from human lungs, and is perfused with microfluidics. Gas and a fluid including culture medium can be perfused into the internal chambers of the system, simulating similar respiratory movements. Even after more than a week following perfusion, the three types of cells within the system can survive. Furthermore, pH and pO2 within the chambers can be monitored using pH and gas partial pressure sensors within the system, allowing the three types of cells to be exposed to the external environment or drugs under conditions similar to those of a living lung. This enables research in a wide range of fields, including modeling lung diseases caused by harmful substances and testing the efficacy of therapeutic drugs. It is also applicable to in vitro disease modeling and customized drug formulation.

Description

Technical Field

[0001] The present invention provides a microfluidic system simulating lung tissue, a method for manufacturing the same, and a microfluidic control method using the microfluidic system. The microfluidic system includes lung epithelial cells, lung fibroblasts, and human umbilical vein endothelial cells isolated from human lungs, and is perfused with microfluidic fluid.

[0002] [National research and development projects supporting this invention]

[0003] [Project Unique Number] 16C1787

[0004] [Department Name] Ministry of Health and Welfare

[0005] [Research and Management Specialist Agency] Korea Health Industry Development Institute

[0006] [Research Project Title] Development of Disease Treatment-Related Technologies

[0007] [Research Topic Title] Clinical Efficacy Study of Novel Drug Candidates for Acute Lung Injury Based on Bionic Chips

[0008] [Contribution Rate] 1 / 1

[0009] [Supervising Institution] Bundang Seoul National University Hospital

[0010] [Research Period] 2016.07.01~2019.08.31. Background Technology

[0011] The lungs are essential organs for respiratory movements, inhaling oxygen and exhaling carbon dioxide through breathing. The alveoli facilitate gas exchange within the lungs. Red blood cells, flowing in the blood through the alveolar capillaries, transport carbon dioxide generated in the body to the lungs, where it is then expelled from the body. Oxygen is inhaled from the air and transported to all parts of the body. Therefore, unlike other organs, the lungs are responsible for gas exchange.

[0012] On the one hand, current in vivo and in vitro models used for lung disease research are limited by the characteristics of the lung and have not been fully developed. For example, animal models do not exhibit the pathological abnormalities of the trachea and lungs frequently found in humans, and most in vitro models cannot simulate the tissue composition and structural complexity of actual tracheal epithelial cells. Furthermore, as mentioned above, in vitro models simulating the lung must be exposed to gases including oxygen for research purposes, but currently commercially available cell-based in vitro models suffer cell death within 3 days when exposed to gases, making them unsuitable for a series of steps requiring at least 7 days, such as model creation, drug efficacy exploration, and drug toxicity screening.

[0013] To address the aforementioned problems, the inventors researched microfluidic systems and completed this invention. The microfluidic system is characterized by the fact that cells can survive for extended periods even during perfusion of gas and fluids including culture medium. Therefore, it enables the simulation of various lung disease models, the testing of therapeutic effects, the testing of other harmful substances, in vitro diagnostics, and customized drug prescriptions. Summary of the Invention

[0014] Technical problems to be solved

[0015] On one hand, the present invention aims to provide a microfluidic system that simulates lung tissue, comprising lung epithelial cells, lung fibroblasts and human umbilical vein endothelial cells isolated from human lungs, and perfused with gas and fluid including culture medium, and including a pH sensor and a sensor for measuring the partial pressure of oxygen (pO2) in the gas.

[0016] On the other hand, the present invention aims to provide a method for manufacturing the microfluidic system that simulates lung tissue.

[0017] In another aspect, the present invention aims to provide a method for monitoring the cell culture environment within a microfluidic system of the simulated lung tissue, the method comprising the steps of perfusing microfluidics into the microfluidic system of the simulated lung tissue and measuring pH and pO2.

[0018] Solution to the problem

[0019] On one hand, the present invention provides a microfluidic system simulating lung tissue, the microfluidic system comprising: a first layer, a second layer, and a third layer; and a first chamber between the first layer and the second layer for perfusion of gas and a second chamber between the second layer and the third layer for perfusion of a fluid including a culture medium; wherein the second layer comprises a porous membrane comprising lung epithelial cells, pulmonary fibroblasts, and human umbilical vein endothelial cells; the lung epithelial cells face the first chamber, the vascular endothelial cells face the second chamber, the pulmonary fibroblasts are present between the vascular endothelial cells and the lung epithelial cells, and the lung epithelial cells and pulmonary fibroblasts are isolated from human lungs, and the first layer and the third layer respectively comprise at least one pH sensor and at least one pO2 sensor.

[0020] On the other hand, the present invention provides a method for manufacturing the microfluidic system simulating lung tissue, the method comprising: (1) coating the second layer of porous membrane with an extracellular matrix; (2) seeding and culturing human lung epithelial cells on the coated porous membrane; and (3) seeding and culturing human fibroblasts and human umbilical vein endothelial cells on the other side of the porous membrane seeded with the lung epithelial cells.

[0021] In another aspect, the present invention provides a method for monitoring the cell culture environment within a microfluidic system for simulating lung tissue, the method comprising: a step of perfusing gas into a first chamber of the microfluidic system for simulating lung tissue and perfusing a fluid comprising culture medium into a second chamber; and a step of measuring pH using a pH sensor of the system and measuring pO2 using a pO2 sensor.

[0022] The effects of the invention

[0023] This invention relates to a microfluidic system simulating lung tissue, comprising lung epithelial cells and lung fibroblasts directly isolated from human lungs. Gas and a fluid including culture medium can be perfused into chambers within the system, and after more than a week of perfusion, all three cell types survive. Furthermore, using pH and pO2 sensors within the system, oxygen transport and pH monitoring can be achieved, thereby determining whether the three cell types are growing under conditions identical to those of a living lung. Therefore, through this microfluidic system simulating lung tissue according to one aspect of the invention, utilizing human lungs (especially those of patients with lung injury), research in a wide range of fields can be achieved, including the creation of lung disease models, efficacy testing of therapeutic drugs, and testing for other harmful substances. Further, it can enable in vitro diagnostics and customized drug formulation. Attached Figure Description

[0024] Figure 1 Photographs of 10 normal lung tissue samples and 2 lung cancer tissue samples used to manufacture a microfluidic system simulating lung tissue according to an embodiment of the present invention.

[0025] Figure 2a A photograph of normal lung epithelial cells (Normal-Epi) dissociated using a method according to an embodiment of the present invention. Figure 2b A photograph of lung cancer cells (cancer-Epi) from lung cancer tissue dissociated using a method according to an embodiment of the present invention.

[0026] Figure 3a This is a FACS analysis result of lung epithelial cells (N-Epi-S1) from normal lung tissue isolated using a method according to an embodiment of the present invention. Figure 3b This is a graph showing the FACS analysis results of lung cancer cells (C-Epi-S1) isolated from lung cancer tissue using a method according to an embodiment of the present invention. Regarding S1 cells obtained from the first patient sample, Epi-S1 was tested using the CD326-PE antibody, indicating successful isolation of cells from both normal and cancerous tissues.

[0027] Figure 4 The image shows the FACS analysis results of lung epithelial cells (N-Epi-S2) and lung fibroblasts (N-fibroblast-S2) from normal lung tissue isolated using a method according to an embodiment of the present invention. S2 cells obtained from a second patient sample were also experimentally analyzed and determined according to the above method.

[0028] Figure 5The image shows the FACS analysis results of lung epithelial cells (N-Epi-S3) and lung fibroblasts (N-fibroblast-S3) from normal lung tissue isolated using a method according to an embodiment of the present invention. S3 cells obtained from a third patient sample were also analyzed and determined according to the above method.

[0029] Figure 6 The image shows the FACS analysis results of lung epithelial cells (N-Epi-S5) and lung fibroblasts (N-fibroblast-S5) isolated from normal lung tissue using a method according to an embodiment of the present invention.

[0030] Figure 7a and Figure 7b This image shows the immunofluorescence analysis results of lung epithelial cells (N-epi-S1 and N-epi-S2) from normal lung tissue isolated using a method according to an embodiment of the present invention. Figure 7c This image shows the immunofluorescence analysis results of small airway epithelial cells (SAECs) from the control group.

[0031] Figure 8 An image showing the results of immunofluorescence analysis of pulmonary fibroblasts (N-Fibroblast-S3) from normal lung tissue isolated using a method according to an embodiment of the present invention.

[0032] Figures 9a to 9c A simplified schematic diagram illustrating the seeding of human lung epithelial cells, lung fibroblasts, and human umbilical vein endothelial cells onto the intermediate porous membrane of a microfluidic system simulating lung tissue according to an embodiment of the present invention. Figure 9a A simplified schematic diagram illustrating the culture results after inoculation with pulmonary fibroblasts and vascular endothelial cells is provided. Figure 9b A simplified schematic diagram illustrating the culture results after inoculation with lung epithelial cells is provided. Figure 9c This is a simplified schematic diagram illustrating the result of the three cell types attaching to the porous membrane of the intermediate layer after inoculation and culture.

[0033] Figure 10a A simplified schematic diagram illustrating the steps of injecting fluid into a microfluidic system simulating lung tissue according to an embodiment of the present invention is provided. Figure 10b A more detailed schematic diagram of the intermediate porous membrane portion of the system, which includes the three types of cells.

[0034] Figure 11 A schematic diagram illustrating the steps of reading actual sensor values ​​in a microfluidic system for simulated lung tissue equipped with a pH sensor and a pO2 sensor according to an embodiment of the present invention.

[0035] Figures 12a to 12d This image shows the results of immunofluorescence analysis after 4 days following perfusion of a fluid including culture medium into a microfluidic system simulating lung tissue according to an embodiment of the present invention.

[0036] Figures 12a to 12d In the diagram, red (Epi) represents lung epithelial cells, and green (Endo) represents vascular endothelial cells. Figure 12a The results were determined by co-culturing epithelial cells and endothelial cells, then staining the epithelial cells red and the endothelial cells green. Figure 12b The results show the lateral appearance of cells observed in ortho mode using confocal microscopy after co-culture. Figure 12c The image shows a cropped section from a three-dimensional video reconstructed from a confocal microscope image using the Imaris program, indicating good co-culture development. Figure 12d To determine from the side Figure 12c The photos were obtained from the video.

[0037] Figure 13a and Figure 13b This image shows the results of immunofluorescence analysis after two days of perfusion of gas and fluid including culture medium into a microfluidic system simulating lung tissue according to an embodiment of the present invention. Figure 13a and Figure 13b In the diagram, green (Fibroblast) represents pulmonary fibroblasts, and red (Epi) represents pulmonary epithelial cells.

[0038] Figures 14a to 14d This image shows the results of immunofluorescence analysis after 4 days following the perfusion of gas and fluid including culture medium into a microfluidic system simulating lung tissue according to an embodiment of the present invention. Figures 14a to 14c In the diagram, red (Epi) represents lung epithelial cells, and green (Endo) represents vascular endothelial cells. Figure 14a The results were determined by co-culturing epithelial cells (Calu3) and endothelial cells (Endo), then staining the epithelial cells red and the endothelial cells green. Figure 14b The results show the lateral appearance of cells observed in ortho mode using confocal microscopy after co-culture. Figure 14c The image shows a cropped section from a three-dimensional video reconstructed from a confocal microscope image using the Imaris program, indicating good co-culture development. Figure 14d To determine from the side Figure 14c The photos were obtained from the video. Figure 14e This is a graph simulating respiratory movements during actual perfusion gas flow. Figure 14f A device used to simulate and measure this respiratory movement.

[0039] Figure 15a and Figure 15b To measure pH using internal pH and pO2 sensors after perfusion fluid is applied to a microfluidic system simulating lung tissue according to an embodiment of the present invention. Figure 15a ) and pO2( Figure 15b The result image. Detailed Implementation

[0040] The present invention will now be described in detail.

[0041] In this invention, "microfluidic" refers to a fine fluid perfused within an individual, specifically the fluid perfused in the human lungs, or more specifically, the gas perfused in the human lungs (e.g., oxygen or carbon dioxide), or the blood, lymph, etc. perfused in the blood vessels that make up the human lungs. In this invention, non-gaseous fluids (such as blood as described above) can be replaced with fluids including culture media in vitro.

[0042] On one hand, the present invention provides a microfluidic system simulating lung tissue, the microfluidic system comprising: a first layer, a second layer, and a third layer; a first chamber for perfusion of gas between the first layer and the second layer, and a second chamber for perfusion of a fluid including a culture medium between the second layer and the third layer; wherein the second layer comprises a porous membrane comprising epithelial cells, fibroblasts, and vascular endothelial cells; the epithelial cells face the first chamber, the vascular endothelial cells face the second chamber, the fibroblasts are present between the vascular endothelial cells and the epithelial cells, the epithelial cells and the fibroblasts are isolated from human lungs, and the first layer and the third layer respectively comprise at least one pH sensor and at least one gas partial pressure sensor.

[0043] According to one aspect of the invention, the microfluidic system simulating lung tissue may include a first layer, a second layer, and a third layer. A first chamber may be included between the first layer and the second layer, and a second chamber may be included between the second layer and the third layer. The first chamber does not include any additional structures constituting the first chamber, but refers to the space formed by the presence of the first and second layers, wherein the first chamber is used for perfusion of gas. Similarly, the second chamber also does not include any additional structures constituting the second chamber, but refers to the space formed by the presence of the second and third layers, wherein the second chamber is used for perfusion of a fluid including a culture medium.

[0044] According to one aspect of the invention, the first layer, the second layer, and the third layer may comprise one or more glasses selected from the group consisting of silicates, borosilicates, and phosphates. Specifically, the first layer, the second layer, and the third layer may each be a borosilicate glass, but are not limited thereto. The substances constituting the first layer, the second layer, and the third layer may be the same as or different from each other.

[0045] According to one aspect of the present invention, the thicknesses of the first layer, the second layer, and the third layer can each be from 0.1 mm to 2 mm, specifically, they can be ≥0.1 mm, ≥0.2 mm, ≥0.3 mm, ≥0.4 mm, ≥0.5 mm, ≥0.6 mm, ≥0.7 mm, ≥0.8 mm, ≥0.9 mm, ≥1.0 mm, ≥1.1 mm, ≥1.2 mm, ≥1.3 mm, ≥1.4 mm, ≥1.5 mm, ≥1.6 mm, ≥1 mm, etc. The thickness can be 0.7mm, ≥1.8mm, or ≥1.9mm, or ≤2.0mm, ≤1.9mm, ≤1.8mm, ≤1.7mm, ≤1.6mm, ≤1.5mm, ≤1.4mm, ≤1.3mm, ≤1.2mm, ≤1.1mm, ≤1.0mm, ≤0.9mm, ≤0.8mm, ≤0.7mm, ≤0.6mm, ≤0.5mm, ≤0.4mm, ≤0.3mm, or ≤0.2mm, but is not limited to these. The thickness of the first layer, the second layer, and the third layer can be the same or different from each other.

[0046] According to one aspect of the invention, the second layer may include a porous membrane. The material constituting the porous membrane may be a polymer, which may be selected from one or more of the group consisting of polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), polycarprolactone (PCL), and nanofibers, but is not limited thereto. The thickness of the porous membrane can be from 3 μm to 24 μm, specifically ≥3 μm, ≥4 μm, ≥6 μm, ≥7 μm, ≥8 μm, ≥9 μm, ≥10 μm, ≥11 μm, ≥12 μm, ≥14 μm, ≥16 μm, ≥18 μm, ≥20 μm or ≥22 μm, or ≤24 μm, ≤22 μm, ≤20 μm, ≤18 μm, ≤16 μm, ≤14 μm, ≤12 μm, ≤11 μm, ≤10 μm, ≤9 μm, ≤8 μm, ≤7 μm, ≤6 μm or ≤4 μm, but is not limited thereto, and can vary with the size of the microfluidic system simulating lung tissue, the second layer or the porous membrane. The pore size of the porous membrane can be from 1 μm to 16 μm, specifically ≥1 μm, ≥2 μm, ≥3 μm, ≥4 μm, ≥5 μm, ≥6 μm, ≥7 μm, ≥8 μm, ≥9 μm, ≥10 μm, ≥11 μm, ≥12 μm, ≥13 μm, ≥14 μm or ≥15 μm, or ≤16 μm, ≤15 μm, ≤14 μm, ≤13 μm, ≤12 μm, ≤11 μm, ≤10 μm, ≤9 μm, ≤8 μm, ≤7 μm, ≤6 μm, ≤5 μm, ≤4 μm, ≤3 μm or ≤2 μm, but is not limited thereto, and can vary with the size of the microfluidic system simulating lung tissue, the second layer or the porous membrane. To enable lung epithelial cells, lung fibroblasts, or vascular endothelial cells to attach to a porous membrane, an extracellular matrix (ECM) can be used to coat the porous membrane. The ECM can be selected from one or more of the following groups: laminin, type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, integrin, nestin, fibronectin, elastin, proteoglycan, hyalin, poly-D-lysine, polysaccharides, and gelatin, but is not limited to these (any substance that can enable lung epithelial cells, lung fibroblasts, or vascular endothelial cells to attach to the porous membrane is acceptable).

[0047] According to one aspect of the invention, the porous membrane may include lung epithelial cells, pulmonary fibroblasts, and human umbilical vein endothelial cells, wherein the lung epithelial cells and the pulmonary fibroblasts may be isolated from human lungs. The lung epithelial cells and pulmonary fibroblasts may be obtained by a separation method comprising: dissociating a lung tissue sample isolated from a human body; adding microbeads specifically binding to the lung epithelial cells and microbeads specifically binding to the pulmonary fibroblasts to the dissociated lung tissue sample; and culturing the lung tissue sample bound to the added microbeads. In this separation method, the microbeads specifically binding to the lung epithelial cells may include cluster of differentiation 326 (CD326), and the microbeads specifically binding to the pulmonary fibroblasts may include anti-fibroblast microbeads. In this context, lung epithelial cells and pulmonary fibroblasts can be isolated separately from human lung tissue samples containing a mixture of various cells. Furthermore, when gas and fluids including culture medium are perfused into a microfluidic system containing simulated lung tissue, including lung epithelial cells, pulmonary fibroblasts, and vascular endothelial cells isolated from human lungs, these cells survive even after 4, 7, or 13 days, unlike single-cell cultures used in existing technologies (Experimental Example 1). Therefore, this enables research in a wide range of fields, such as testing the efficacy of drugs for treating lung diseases and testing for other harmful substances. It can also be further applied to in vitro diagnostics and customized drug formulation.

[0048] According to one aspect of the invention, the lung epithelial cells may face the first chamber, the vascular endothelial cells may face the second chamber, and the pulmonary fibroblasts may be present between the lung epithelial cells and the vascular endothelial cells. Based on the positions of the lung epithelial cells and vascular endothelial cells as described above, the lung epithelial cells may be in communication with the gas perfused in the first chamber, and the vascular endothelial cells may be in communication with the fluid including culture medium perfused in the second chamber, thus existing in a microenvironment similar to that of an actual human lung.

[0049] According to one aspect of the invention, the lung epithelial cells may be alveolar epithelial cells, which may include type I alveolar epithelial cells and type II alveolar epithelial cells. The ratio of type I alveolar epithelial cells to type II alveolar epithelial cells may be from 7:3 to 9:1, but is not limited thereto.

[0050] According to one aspect of the invention, the first layer may include a first injection port for injecting gas, a first outlet port for discharging the gas, a second injection port for injecting fluid comprising culture medium, and a second outlet port for discharging the fluid, and the second layer may include two or more channels for perfusing fluid comprising culture medium from the first chamber into the second chamber. There may be one or more of these first injection ports, first outlet ports, second injection ports, and second outlet ports.

[0051] According to one aspect of the invention, the first layer and the third layer respectively include at least one pH measuring sensor and at least one gas partial pressure measuring sensor. Specifically, the first layer may include at least one pH measuring sensor or at least one gas partial pressure measuring sensor, and the third layer may include at least one pH measuring sensor or at least one gas partial pressure measuring sensor. More specifically, the first layer may include at least one gas partial pressure measuring sensor, and the third layer may include at least one pH measuring sensor. These pH measuring sensors may be sensors for measuring the pH inside the microfluidic system of the simulated lung tissue, or sensors for measuring the pH in a second chamber perfused with a fluid including culture medium injected from the second inlet. These gas partial pressure measuring sensors may be sensors for measuring the partial pressure of oxygen (pO2), or sensors for measuring the partial pressure of oxygen in a first chamber perfused with gas injected from the first inlet. These pH measuring sensors may be sensors for real-time pH measurement, and these gas partial pressure measuring sensors may be sensors for real-time gas partial pressure measurement.

[0052] According to one aspect of the invention, the microfluidic system simulating lung tissue can be used to measure oxygen partial pressure and pH in real time, thereby adjusting the amount of oxygen transported into the system and the pH inside the system (Experimental Example 3).

[0053] On the other hand, the present invention provides a method for manufacturing the microfluidic system of the simulated lung tissue. The method comprises: (1) coating the second layer of the porous membrane with an extracellular matrix; (2) seeding and culturing human-derived pulmonary fibroblasts and endothelial cells on the coated porous membrane; and (3) seeding and culturing human-derived lung epithelial cells on the other side of the porous membrane seeded with the pulmonary fibroblasts and endothelial cells. The microfluidic system of the simulated lung tissue, the second layer, the porous membrane, the extracellular matrix, the lung epithelial cells, the pulmonary fibroblasts, and the endothelial cells are described above.

[0054] According to one aspect of the present invention, the extracellular matrix in step (1) described above may be selected from one or more of the group consisting of laminin, type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, integrin, nestin, fibronectin, elastin, proteoglycan, hyalin, poly-D-lysine, polysaccharides, and gelatin, but is not limited thereto (any substance that can enable lung epithelial cells, lung fibroblasts, or vascular endothelial cells to attach to a porous membrane is acceptable).

[0055] According to one aspect of the invention, the lung epithelial cells and the lung fibroblasts can be isolated from human lungs, specifically obtained by the following separation method, which includes: dissociating a lung tissue sample isolated from a human body; adding microbeads specifically bound to the lung epithelial cells and microbeads specifically bound to the lung fibroblasts to the dissociated lung tissue sample; and culturing the lung tissue sample bound to the added microbeads. In this separation method, the microbeads specifically bound to the lung epithelial cells may include cluster of differentiation 326 (CD326), and the microbeads specifically bound to the lung fibroblasts may include anti-fibroblast microbeads. In this case, lung epithelial cells and lung fibroblasts can be separated by type from a human lung tissue sample containing a mixture of various cells. Lung tissue samples bound to these microbeads can be cultured at temperatures ranging from 4°C to 37°C. Specifically, the temperatures can be ≥4°C, ≥5°C, ≥10°C, ≥15°C, ≥20°C, ≥25°C, ≥30°C, or ≥35°C, or ≤37°C, ≤35°C, ≤30°C, ≤25°C, ≤20°C, ≤15°C, ≤10°C, ≤5°C, or ≤4°C, but are not limited to these ranges (the culture temperature may vary depending on the type of lung epithelial cells and pulmonary fibroblasts). Furthermore, when gas and fluids including culture medium are perfused into a microfluidic system containing simulated lung tissue, including lung epithelial cells, pulmonary fibroblasts, and vascular endothelial cells isolated from human lungs, these cells survive even after 4, 7, or 13 days, unlike single-cell cultures used in existing technologies (Experimental Example 1). Therefore, this enables research in a wide range of fields, such as testing the efficacy of drugs for treating lung diseases and testing for other harmful substances. It can also be used for in vitro diagnostics and customized drug formulation.

[0056] According to one aspect of the present invention, the mixing ratio of pulmonary fibroblasts and vascular endothelial cells in step (3) described above can be from 1:1 to 1:25, specifically, it can be ≥1:1, ≥1:2, ≥1:3, ≥1:4, ≥1:5, ≥1:6, ≥1:8, ≥1:10, ≥1:15 or ≥1:20, or it can be ≤1:25, ≤1:20, ≤1:15, ≤1:10, ≤1:8, ≤1:6, ≤1:5, ≤1:4, ≤1:3 or ≤1:2, but is not limited thereto.

[0057] In another aspect, the present invention provides a method for controlling microfluidics within a microfluidic system simulating lung tissue. The method includes a microfluidic perfusion step of perfusing gas into a first chamber of the microfluidic system simulating lung tissue and perfusing a fluid comprising culture medium into a second chamber; and a measurement step of measuring pH using a pH sensor of the system and measuring gas partial pressure using a gas partial pressure sensor. The microfluidic system simulating lung tissue, the first chamber, the second chamber, the gas, the fluid comprising culture medium, pH measurement, and gas partial pressure measurement are described above.

[0058] According to one aspect of the invention, the control method further includes: adjusting the hourly injection volume of gas and fluid including culture medium into the microfluidic system of the simulated lung tissue when the pH or gas partial pressure measured in the pH and gas partial pressure measurement step is different from the pH or gas partial pressure in human lung tissue.

[0059] According to one aspect of the invention, the gas partial pressure may be the oxygen partial pressure (pO2).

[0060] The structure and effects of the present invention will be described in more detail below with reference to embodiments and experimental examples. However, the following embodiments and experimental examples are merely illustrative and provided to help better understand the present invention, and are not intended to limit the scope and range of the present invention.

[0061] Example 1: Isolation of lung epithelial cells and pulmonary fibroblasts from human lung tissue

[0062] To create a microfluidic system that simulates living lung tissue, lung epithelial cells and lung fibroblasts were isolated from human lung tissue using the following method.

[0063] Example 1-1 Dissociation of human lung tissue

[0064] Ten normal lung tissue samples (N-1 to N-10) ranging from 0.39 g to 1.37 g, obtained from normal lung tissue in resected lung tissue of lung cancer patients who had undergone surgery, and two lung cancer tissue samples (C-1 and C-2) ranging from 0.41 g, obtained from cancerous tissue in resected lung tissue of lung cancer patients, were stored in storage buffer (Miltenyi Biotec, Tissue Storage Solution) at 4°C for 1 to 2 days. These tissue samples were then aliquoted into 0.4 g to 0.5 g portions and placed into individual C tubes (C test tubes).

[0065] Table 1

[0066] Tissue sample number Weight (g) Tissue sample number Weight (g) N-1 0.54 N-7 1.37 N-2 0.52 N-8 0.64 N-3 0.66 N-9 1.00 N-4 0.70 N-10 0.39 N-5 1.02 <![CDATA[C-1 * ]]> 0.28 N-6 1.17 <![CDATA[C-2 * ]]> 0.13

[0067] *The total weight of lung cancer tissue samples C-1 and C-2 is 0.41g. Using these lung cancer tissue samples, lung cancer cells were isolated from the lung cancer tissue using the following method.

[0068] Next, add 2.35 ml of serum-free culture medium—RPMI medium (Welgene, RPMI 1640)—to each C-tube segment. Following the instructions, add the enzyme kit (Miltenyi Biotec, Multi Tissue Dissociation Kit I) to each C-tube, wash, and remove excess water from the washed tissue samples. Then, place each C-tube into a separate C-tube. Next, insert scissors into the inside of each C-tube to cut the tissue into smaller pieces as much as possible, and cap the tube. Then, insert each C-tube containing ice into a MACS (Magnetically Activated Cell Sorting) device (Miltenyi Biotec, GentleMACS) in sequence. TM Run the program (Octo Dissociator). If an error occurs, remove the C test tubes, mix them, and rerun the program. After the program finishes, remove the C test tubes with ice cubes inserted.

[0069] Install a filter on a 15ml or 50ml test tube, moisten the filter with approximately 2ml of serum-free RPMI culture medium, and then at the end of the procedure, pour the lung tissue sample included in each C tube into these filters, and rinse the C tubes again with 5ml of the same serum-free RPMI culture medium before pouring the solution into the filters. Then, centrifuge at 300xg for 10 minutes, remove the supernatant, add 1ml of red blood cell lysis buffer (Lonza), let stand for approximately 2 minutes, and then resuspend the sample in 5ml of serum-free RPMI culture medium. Then, the cells were centrifuged at 300xg for 10 minutes, the supernatant was removed, and the cells were resuspended in 5ml to 10ml of cell culture medium (Lonza, EGM-2). The cell count was then calculated, and the cells were placed back into a T-175 flask containing endothelial cell culture medium (Lonza, EGM-2) or epithelial cell culture medium (Lonza, SAGM) for further culture, thus detaching the human lung tissue. At this point, each C tube was placed into a T-175 flask for culture.

[0070] In the dissected lung tissue, after observing the T-175 flasks containing cultured cells using an optical microscope connected to a camera, photographs of lung epithelial cells and lung fibroblasts were taken. The results are as follows: Figure 2a (Lung epithelial cells isolated from normal lung tissue samples (Normal-Epi)) and Figure 2b (Lung cancer cells (cancer-Epi) isolated from a lung cancer tissue sample) is shown.

[0071] Examples 1-2: Isolation of lung epithelial cells and lung fibroblasts

[0072] In Example 1-1, the dissociated human lung tissue was separated into lung epithelial cells and lung fibroblasts through the steps described below.

[0073] Specifically, the number of cells contained in the T-175 flask in Example 1-1 was calculated, and 5 x 10⁻⁶ cells were prepared. 6 Up to 1X10 7After cell separation, the cells were centrifuged at 300 x g for 10 minutes at room temperature to prepare suspensions of three cell clumps: a total of 300 μL of lung epithelial cell suspension and a total of 80 μL of lung fibroblast suspension. At this point, the three suspensions were prepared using PEB buffer, obtained by mixing 47.5 mL of autoMACS rinsing solution (Miltenyi Biotec, #130-091-222) and MACS BSA stock solution (Miltenyi Biotec, #130-091-376) at a volume ratio of 20:1.

[0074] Then, in order to improve the purity of the isolated cells, 100 μL of FcR blocking reagent (Miltenyi Biotec, #130-059-901) was added to the lung epithelial cell suspension, while no FcR blocking reagent was added to the lung fibroblast suspension.

[0075] Then, 100 μL of CD326 microbeads (Miltenyi Biotec, #130-061-101) were added to the lung epithelial cell suspension, and 20 μL of anti-fibroblast microbeads (Miltenyi Biotec, #130-050-601) were added to the lung fibroblast suspension. The suspensions were then thoroughly mixed by shaking with Vertex gel. The mixture of CD326 microbeads and lung epithelial cell suspension was cultured at 4°C for approximately 30 minutes, and then at room temperature for approximately 30 minutes. All cultures were maintained in a cold storage facility. After the above culture, 1 ml of PEB buffer was added to the mixture of CD326 microbeads and lung epithelial cell suspension for washing, and then centrifuged at 300 x g for 10 minutes. Similarly, 1 ml of PEB buffer was added to the mixture of anti-fibroblast microbeads and lung fibroblast suspension for washing, and then centrifuged at 300 x g for 3 minutes. Then, 1 ml of PEB buffer was added to each mixture to prepare two suspensions: a lung epithelial cell suspension bound to CD326 microbeads and a lung fibroblast suspension bound to anti-fibroblast microbeads.

[0076] In these suspensions, to utilize the magnetic properties of the microbeads for cell separation, an LS column is placed inside a cell separator (Mitenyi Biotec, MidiMACS).TM In the magnetic separator, rinse the sorting column with 3 ml of PEB buffer. Then, place a 15 ml tube at the bottom of the sorting column, and allow the suspension of CD326 microbead-bound lung epithelial cells to flow into the sorting column. Collect the unlabeled negative cells (unlabeled cells) in the 15 ml tube. Add 3 ml of PEB buffer to the sorting column on ice. If all the buffer flows out, add another 3 ml of PEB buffer. Repeat this step 3 times for washing. To collect the microbead-labeled cells, remove the sorting column from the cell separator and place a new 15 ml tube at the top. Then, add 5 ml of PEB buffer to the sorting column and press the plunger until bubbles appear. Collect the CD326 microbead-bound and labeled lung epithelial cells (labeled cells) in the 15 ml tube at the bottom of the sorting column. After centrifuging at 300xg for 10 minutes, the supernatant was removed, and 10ml of cell culture medium was resuspended. The number of lung epithelial cells was then counted. Labeled lung fibroblasts were collected from the suspension of anti-fibroblast microbeads using the microbead magnetic cell separation method described above. The cells were then centrifuged, resuspended, and counted in the same manner.

[0077] Examples 1-3 use FACS analysis to determine the separation of lung epithelial cells and lung fibroblasts.

[0078] To determine whether the lung epithelial cells and lung fibroblasts obtained from Examples 1-2 were separated by species, analysis was performed using a fluorescence activated cell sorter (FACS).

[0079] Specifically, PEB buffer was prepared by mixing 47.5 ml of autoMACS rinsing solution (Miltenyi Biotec, #130-091-222) and MACS BSA stock solution (Miltenyi Biotec, #130-091-376) at a volume ratio of 20:1. Using trysin / EDTA (Welgene, LS015-01), lung epithelial cells and pulmonary fibroblasts isolated in Examples 1-2 were labeled S1 (first) and S2 (second) according to the order in which patient samples were obtained. Normal cells were labeled N, and cancer cells were labeled C. N1 to C2 are the classification numbers after the two types of tissues were cut into 0.5-gram units. Each type of cell was counted separately, and 2 x 10⁻⁶ cells were collected from each cell type. 5 Then, centrifuge at 300 x g for 10 minutes. The FACS analysis results are shown in Figures 3 to 4. Figure 6 As shown.

[0080] As shown in Figure 3 to Figure 6 As shown, according to the FACS histogram results, the black line represents the control group without attached antibodies, and the lines of other colors represent the cell count of the experimental group with attached antibodies. By confirming that there is a significant difference between the black lines of the experimental group and the control group, it can be determined that the separation is good.

[0081] Examples 1-4 used immunofluorescence assays to determine the separation of lung epithelial cells and lung fibroblasts.

[0082] To determine whether the lung epithelial cells and lung fibroblasts obtained from Examples 1-2 were isolated by species, an immunofluorescence assay was performed.

[0083] (1) The isolation of lung epithelial cells was determined by immunofluorescence analysis.

[0084] First, in Examples 1-2, after 3 days of isolating lung epithelial cells, N-epi-S1 was reacted with the epithelial cell marker, mouse mAb anti-E cadherin (Santa Cruz, sc-8426) (Santa Cruz, sc-73480 ... Figure 7a As shown. Additionally, the N-epi-S2 lung epithelial cells isolated in Examples 1-2 were also analyzed by immunofluorescence using the same method as described above, but with the difference that: the mouse monoclonal antibody anti-E cadherin (Abcam, ab1416) was used instead of the epithelial cell marker—the mouse monoclonal antibody anti-E cadherin (Santa Cruz, sc-8426)—as the primary antibody, and the reaction was performed at a dilution of 1:50; and donkey anti-mouse IgG-Alexa 488 (Abcam, ab150105) was used instead of goat anti-mouse IgG-Alexa 488 (MP, A111001) as the secondary antibody, and the reaction was performed at a dilution of 1:100. Images were captured using a confocal microscope at X200 magnification. The results for N-epi-S2 are shown below. Figure 7b As shown.

[0085] On the one hand, as a control group, small airway epithelial cells (SAEC) (Lonza, CC-2547) were analyzed for immunofluorescence using the same method as the N-epi-S2 immunofluorescence assay. The results for the control group were as follows: Figure 7c As shown.

[0086] like Figure 7a and Figure 7b As shown, the control group ( Figure 7c The commercially available epithelial cells used expressed E-calcium adhesion protein and cytokeratin 8. Similarly, it was determined that the lung epithelial cells actually isolated from humans in Examples 1-2 also expressed E-calcium adhesion protein and cytokeratin 8.

[0087] (2) Immunofluorescence analysis was used to determine the isolation of pulmonary fibroblasts.

[0088] First, in Examples 1-2, after isolating pulmonary fibroblasts for 3 days, N-Fibroblast-S3 was reacted with the epithelial cell marker as the primary antibody—mouse mAb anti-ECadherin (Santa Cruz, sc-8426) at a ratio of 1:100, the endothelial cell marker—mouse mAb anti-CD31 (Santa Cruz, sc-65260) at a ratio of 1:100, and the fibroblast marker—mouse mAb anti-αSmooth Muscle Actin (Abcam, ab7817) at a ratio of 1:200. Then, it was reacted with the secondary antibody—goat anti-mouse IgG-Alexa 488 (MP, A111001) at a ratio of 1:100. The reaction was then performed using a confocal microscope (Carl). The images were taken with a Zeiss LSM710, and the results are as follows: Figure 8 As shown.

[0089] like Figure 8 As shown, it was determined that the lung fibroblasts actually isolated from the human body in Examples 1-2 expressed only α-smooth muscle actin.

[0090] Based on the results of Examples 1-3 and 1-4, it was determined that lung epithelial cells and lung fibroblasts could be isolated from actual human subjects via the steps of Examples 1-1 and 1-2.

[0091] Example 2: Fabrication of a microfluidic system comprising lung epithelial cells, pulmonary fibroblasts, and human umbilical vein endothelial cells isolated from human lung tissue to simulate lung tissue.

[0092] Using lung epithelial cells, lung fibroblasts, and human umbilical vein endothelial cells isolated from human lung tissue obtained in Example 1, a microfluidic system simulating in vivo lung tissue was fabricated by the method described below. Alternatively, Calu-3 (ATCC) can also be used in addition to the lung epithelial cells obtained in Example 1.

[0093] First, a mixture of 0.001% fibronectin (Sigma, F0895, 5 mg), 0.03 mg / ml collagen (Sigma, Collagen Type I solution, 1 VL content), and 0.001 mg / ml bovine serum albumin (BSA) (Miltenyi Biotec, MACS BSAstock #130-091-376, 75 ml) was coated at 4°C onto a porous membrane (pore size 0.45 μm, pore density 2.00) in the middle layer of an organ-on-a-chip platform (Micronit, Netherlands). 6 cm 2 When the pore size is 3μm, the pore density is 8.00. 5 cm 2 After one night, allow it to dry. Then, use the 2 x 10 isolates obtained from the human body in Example 1. 5 / 100μl of lung epithelial cells (N-Epi-S2 (p3: passaged 3 times) or Calu-3) were seeded on the lower end face of the porous membrane at 37°C for about 2 hours, and then cultured at 37°C for 1 day. Figure 9a Then, 2 x 10 5 / 50μl of human umbilical vein endothelial cells (HUVEC(p2), LONZA) and 0.4 x 10⁻⁶ human cells isolated in Example 1. 5 A mixture of 50 μl of lung fibroblasts (N-Fibroblast-S5(p8)) was seeded onto the other side of the porous membrane, opposite the side seeded with lung epithelial cells, and cultured at 37°C for 4 days. Figure 9b ). Figure 9c A simplified schematic diagram showing the attachment of lung epithelial cells, vascular endothelial cells, and pulmonary fibroblasts to a porous membrane.

[0094] According to the organ-on-a-chip platform's specifications, the microfluidic system simulating lung tissue is manufactured to include an intermediate layer comprising lung epithelial cells, vascular endothelial cells, and lung fibroblasts. Figure 10a This is a simplified schematic diagram of the microfluidic system for simulating lung tissue. Figure 10b A simplified schematic diagram of the porous membrane in the system, which includes the intermediate layer of these three types of cells.

[0095] Additionally, a pO2 sensor (Presens, Microox4) serving as a gas partial pressure measuring sensor is installed on the side of the first chamber (for perfusion gas) facing the first layer (or upper layer) of the system, and a pH measuring sensor (Presens, pH-1mini V2) is installed on the side of the second chamber (for perfusion of fluid including culture medium) facing the third layer (or lower layer). Figure 11 This is a simplified schematic diagram of a microfluidic system for simulating lung tissue equipped with the aforementioned sensors.

[0096] Example 3: Perfusion fluid in a microfluidic system simulating lung tissue

[0097] Gas (air flowing through the system connected to the gas pump, gas sensor, and tubing, and composed of actual atmospheric air at room temperature) is injected into the first inlet of the first layer (or upper layer) of the microfluidic system for simulating lung tissue manufactured in Example 2, and culture medium (STEMCELL, PneumaCult) is injected into the second inlet. TM Following the administration of ALI#05001 (500ml), perfusion was carried out at a rate of 5μl / min for 4 days to create a microfluidic system similar to that of living lung tissue. Additionally, the pressure range was adjusted through a series of steps to achieve a perfusion gas cycle of 10-12 breaths / min, simulating actual respiratory movements. Figure 14e and 14f ).

[0098] Experiment 1: Determining cell viability in a microfluidic system simulating lung tissue

[0099] This study aimed to determine the viability of lung epithelial cells, pulmonary fibroblasts, and vascular endothelial cells in a microfluidic system simulating lung tissue manufactured according to Example 2, after perfusion fluid as described in Example 3 and for several days. To determine cell survival after culture, LIVE / DEAD was used in this experiment. TM The viability / cytotoxicity kit (Invitrogen, #L3224) was used to stain surviving and dead cells at 37°C for 30 minutes, and then the viability was determined using a confocal microscope (Carl Zeiss, LSM710).

[0100] Figures 12a to 12d The immunofluorescence analysis results of the perfusion fluid after 4 days confirmed that the lung epithelial cells, lung fibroblasts and vascular endothelial cells in the microfluidic system simulating lung tissue were all alive after 4 days.

[0101] in addition, Figures 13a to 13b After 2 days of perfusion fluid and Figures 14a to 14dThe immunofluorescence analysis results of the perfusion fluid after 4 days confirmed that the lung epithelial cells, lung fibroblasts and vascular endothelial cells in the microfluidic system simulating lung tissue were also alive after 2 days and 4 days.

[0102] It is hereby stated that the above Figures 12a to 12d , Figure 13a and Figure 13b In order to utilize the lung epithelial cells obtained from Example 1, Figures 14a to 14d For the case of utilizing Calu-3.

[0103] Experiment 2 determined oxygen transport and pH regulation in a microfluidic system simulating lung tissue.

[0104] Oxygen transport and pH regulation are the most important and fundamental functions of the lungs. To determine these functions, in the microfluidic system simulating lung tissue manufactured in Example 2, oxygen partial pressure and pH were measured after perfusion fluid was applied as described in Example 3. The pH sensor measurement procedure is as follows: A pH sensor is mounted inside the glass chip at both the upper and lower ends. During the cell culture step, the fiber optic cable of a pH-1 mini V2 transmitter, connected to a computer for monitoring, is brought into contact with the sensor from the outside to measure pH in real time. The O2 sensor measurement procedure is as follows: An O2 sensor is mounted inside the glass chip at both the upper and lower ends. During the cell culture step, the fiber optic cable of a pO2 Microox4 transmitter is brought into contact with the sensor from the outside to measure pO2 in real time.

[0105] Figure 15a and Figure 15b To measure pH using internal pH and pO2 sensors after perfusion fluid is applied to a microfluidic system simulating lung tissue according to an embodiment of the present invention. Figure 15a ) and pO2( Figure 15b The result image.

[0106] like Figure 15a and Figure 15b As shown, the oxygen partial pressure inside the system is equivalent to 20% of the oxygen partial pressure in the atmosphere, and the pH is also the same as the pH of the injected cell culture medium, which is 7.0 to 8.0.

[0107] That is, by using the microfluidic system for simulating lung tissue manufactured in Example 2, the partial pressure of oxygen and pH can be measured in real time, thereby adjusting the amount of oxygen transported into the system and the pH inside the system.

[0108] Industrial utilization

[0109] The microfluidic system for simulating lung tissue provided by one aspect of the present invention can realize research in a wide range of fields, including the realization of lung disease models, efficacy testing of therapeutic drugs, and testing of other harmful substances. It can also be applied to in vitro diagnostics and customized drug prescriptions.

Claims

1. A microfluidic system simulating lung tissue, comprising: The first layer, the second layer, and the third layer; and a first chamber for perfusion gas between the first layer and the second layer; and a second chamber between the second and third layers for perfusing fluid including culture medium; The second layer includes a porous membrane; Lung epithelial cells were inoculated and cultured on one side of the porous membrane, and a mixture of pulmonary fibroblasts and vascular endothelial cells was inoculated and cultured on the other side of the porous membrane where the lung epithelial cells were inoculated and cultured. The lung epithelial cells face the first chamber, and the vascular endothelial cells face the second chamber; The lung epithelial cells and the lung fibroblasts were isolated from human lungs; The lung epithelial cells are alveolar epithelial cells; and The mixing ratio of the pulmonary fibroblasts and the vascular endothelial cells is 1:4 to 1:6; The first and third layers contain one or more pH sensors and / or one or more gas partial pressure sensors; The pH sensor is a sensor that measures pH in real time; and The gas partial pressure measuring sensor is a sensor that measures the partial pressure of gases in real time.

2. The microfluidic system for simulating lung tissue according to claim 1, wherein: The lung epithelial cells include type I alveolar epithelial cells and type II alveolar epithelial cells, and the ratio of type I alveolar epithelial cells to type II alveolar epithelial cells is 7:3 to 9:

1.

3. The microfluidic system for simulating lung tissue according to claim 1, wherein: At least a portion of the system is transparent.

4. A microfluidic system for simulating lung tissue according to claim 1, wherein: The first layer includes a first injection port for injecting gas, a first outlet port for discharging the gas, a second injection port for injecting a fluid including culture medium, and a second outlet port for discharging the fluid; The second layer includes two or more channels for perfusing the fluid, including the culture medium, from the first chamber into the second chamber.

5. A microfluidic system for simulating lung tissue according to claim 1, wherein: The porous membrane is coated with an extracellular matrix.

6. A microfluidic system for simulating lung tissue according to claim 5, wherein: The extracellular matrix may be selected from one or more of the following groups: laminin, type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, integrin, nestin, fibronectin, elastin, proteoglycan, hyalin, poly-D-lysine, polysaccharides, gelatin, or matrix adhesive.

7. A microfluidic system for simulating lung tissue according to claim 1, wherein: The pore size of the porous membrane is 0.45 μm or 3 μm.

8. A microfluidic system for simulating lung tissue according to claim 1, wherein: The lung epithelial cells are in communication with the gas injected from the first injection port.

9. A microfluidic system for simulating lung tissue according to claim 1, wherein: The vascular endothelial cells are in fluid communication with a culture medium injected from the second inlet.

10. A method for manufacturing a microfluidic system simulating lung tissue according to any one of claims 1 to 9, comprising: (1) The step of coating the porous membrane of the second layer with an extracellular matrix; (2) The step of inoculating and culturing human lung epithelial cells on the coated porous membrane; and (3) The step of inoculating and culturing pulmonary fibroblasts and vascular endothelial cells isolated from the human body on the other side of the porous membrane inoculated with the lung epithelial cells.

11. A method for manufacturing the microfluidic system for the simulated lung tissue according to claim 10, wherein: The extracellular matrix in step (1) may be selected from one or more of the following groups: laminin, type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, integrin, nestin, fibronectin, elastin, proteoglycan, hyalin, poly-D-lysine, polysaccharides, gelatin, or matrix adhesive.

12. A method for manufacturing the microfluidic system for the simulated lung tissue according to claim 10, wherein: The lung epithelial cells and lung fibroblasts were obtained according to the following steps: The steps for dissociating lung tissue samples isolated from the human body; The steps of adding microbeads specifically binding to the lung epithelial cells and microbeads specifically binding to the lung fibroblasts to the dissociated lung tissue sample; and The step of culturing lung tissue samples combined with the added microbeads.

13. A method for manufacturing the microfluidic system for the simulated lung tissue according to claim 12, wherein: The microbeads that specifically bind to the lung epithelial cells include differentiation clusters 326; The microbeads that specifically bind to the pulmonary fibroblasts include anti-fibroblast microbeads.

14. A method for manufacturing the microfluidic system for the simulated lung tissue according to claim 12, wherein: Lung tissue samples bound to the microbeads were cultured at an environment of 4°C to 25°C.

15. A method for controlling a microfluidic system simulating lung tissue, comprising: The microfluidic perfusion step of perfusing gas into the first chamber of the microfluidic system for simulating lung tissue according to claim 1, and perfusing a fluid including culture medium into the second chamber; The steps for adjusting the perfusion gas pressure to simulate respiratory movements; as well as The measurement steps include using the pH sensor of the system to measure pH and using the gas partial pressure sensor to measure gas partial pressure.

16. A method for controlling the microfluidic system of the simulated lung tissue according to claim 15, wherein: The control method further includes the step of adjusting the hourly injection volume of the gas injected into the microfluidic system of the simulated lung tissue and the fluid including the culture medium when the pH or gas partial pressure measured in the pH and gas partial pressure measurement step is different from the pH or gas partial pressure of human lung tissue.

17. A method for controlling the microfluidic system of the simulated lung tissue according to claim 15, wherein: The partial pressure of the gas is the partial pressure of oxygen, pO2.

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