Lung bronchiole-microenvironment-mimetic biological tissue chip

Abstract

The present invention provides a lung bronchiole-microenvironment-mimetic biological tissue chip as a platform for testing the inhalation of chemical harmful substance, respiratory toxicity, and pulmonary disease drug efficacy.

Description

Biotissue chip mimicking the pulmonary bronchial microenvironment

[0001] The present invention relates to a bio-tissue chip that mimics the microenvironment of a pulmonary bronchiole, and more specifically, to a bio-tissue chip that mimics the microenvironment of a pulmonary bronchiole as a platform for testing inhalation of chemical hazardous substances, respiratory toxicity, and efficacy of lung disease drugs.

[0002] Drug development requires significant time, effort, and expense, yet only a small fraction of those in development receive approval. Achieving regulatory approval for a single drug can be prohibitively expensive, costing upwards of $2.5 billion and taking a long time, typically 10-15 years. To overcome this, numerous animal or cell-based experiments have been routinely conducted in the early stages of drug development.

[0003] Animal-based experiments are expensive, time-consuming, and inherently ethical. Furthermore, they suffer from limitations such as the presence of different ion channels and pharmacokinetic differences between humans and other species. Consequently, animal test results often do not apply to humans. Even if a drug passes clinical trials after completing animal testing, it may be withdrawn due to adverse effects. In cell-based experiments, cells are typically cultured in two-dimensional (2D) conditions. While these 2D cell culture models offer the advantages of ease of handling and high throughput, their use in drug efficacy and toxicity assessments is limited because cells lose their inherent functions during the 2D culture process, leading to erroneous results. In human organs, cells exist in a three-dimensional (3D) environment with the extracellular matrix (ECM). They receive nutrients and oxygen through diffusion from capillaries, while waste products, metabolites, and carbon dioxide are excreted through the blood vessels. Therefore, an efficient drug efficacy and toxicity assessment platform that reflects the structural, physiological, and environmental characteristics of actual organs is needed.

[0004] A biotissue-on-a-chip (OoC), also known as a biomimetic chip or organ-on-a-chip (OoC), is a system that mimics the internal microenvironment of a specific human organ and cultures cells constituting that organ in a microchamber to replicate its characteristics. Therefore, OoC represents an effective alternative that can overcome the limitations of animal testing and, by realizing physiological activity at the tissue or organ level, holds promise as a platform for preclinical new drug development.

[0005] Recent drug efficacy and toxicity assessments are focusing on the pharmacodynamic effects of multi-organ interactions. Multi-organs-on-chip research is being attempted, but it remains in its infancy. Most of these studies simply co-culture cells from multiple organs in two dimensions, without reproducing the core microstructure of each organ, to assess the effectiveness of the circulatory system. To overcome this limitation, the development of a system capable of co-culturing two or more organs by reproducing the key microstructures of each organ is necessary. Therefore, the development of innovative multi-organ-on-chips that mimic the core microstructure of each organ and reflect the pharmacodynamic effects of drug interactions across organs is essential.

[0006] Currently, numerous types of chemical substances are being developed in the chemical industry, and among these, the number of chemical substances that can be exposed to the respiratory system through chemical products and over-the-counter drugs is increasing. Accordingly, as cases of damage caused by inhaled chemicals are emerging as a social problem, the importance of inhalation toxicity tests for safety evaluation is increasing.

[0007] Currently, in chemical safety management, acute inhalation toxicity is classified into 4 stages according to the degree of toxicity with reference to the UN GHS (United Nations Global Harmonization System), and the acute inhalation toxicity test method for this classification is based on animal testing in OECD Test Guideline 403. The current acute inhalation toxicity test method (OECD TG403) requires a large number of animals, high cost, and long time, so interest in developing alternative test methods that can replace it is increasing. However, there is no internationally verified or recognized alternative test method for inhalation toxicity, so it is currently not possible to replace animal testing on its own.

[0008] Accordingly, there is an increasing need for the development of an integrated inhalation toxicity prediction test method that can maximize predictive power by advancing and integrating the latest technologies, such as human-derived 3D lung cell culture methods, lung microphysiological systems (MPS), and in silico technologies, which are currently being actively developed.

[0009] The technical problem to be solved by the present invention is to provide a bio-tissue chip that mimics the microenvironment of a pulmonary bronchiole, which can individually and simultaneously culture cells derived from different organs, as a platform for testing the inhalation of chemical hazardous substances, respiratory toxicity, and the efficacy of lung disease drugs.

[0010] The technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0011] According to one embodiment of the present invention for achieving the above technical task, a pulmonary bronchial microenvironment-mimicking bio-tissue chip is a pulmonary bronchial microenvironment-mimicking bio-tissue chip that cultures fixed pulmonary epithelial cells by supplying a culture medium to the fixed pulmonary epithelial cells, wherein the bio-tissue chip includes a culture medium channel, an extracellular matrix channel, and a cell chamber, the pulmonary epithelial cells are fixed to the cell chamber, and the culture medium is supplied from the culture medium channel through the extracellular matrix channel to the cell chamber.

[0012] The above extracellular matrix channel can form a hydrogel inside.

[0013] At this time, the flow rate of the culture medium may be reduced during the process of being supplied from the culture medium channel to the cell chamber through the extracellular matrix channel.

[0014] Additionally, the extracellular matrix channel can further culture pulmonary fibroblasts inside it.

[0015] The above culture medium channel can further culture vascular endothelial cells inside.

[0016] The above lung epithelial cells can be fixed in the cell chamber by mixing with any one of a hydrogel, a collagen hydrogel, a fibronectin hydrogel, or a lastin hydrogel.

[0017] The above biotissue chip includes a mixing region in which the culture medium can interact with each other, and in the mixing region, the culture medium channel, the extracellular matrix channel, and the cell chamber can be arranged in parallel in the longitudinal direction.

[0018] At this time, the width of the cell chamber may be wider than the width of the culture medium channel, and the width of the culture medium channel may be wider than the width of the extracellular matrix channel.

[0019] The above-mentioned biotissue chip may further include a first-stage micro-weave structure having a space separated from the bottom at the bottom, and the first-stage micro-weave structure may be formed at an adjacent portion between the culture medium channel and the extracellular matrix channel and an adjacent portion between the extracellular matrix channel and the cell chamber, respectively.

[0020] At this time, the culture medium can be moved to the extracellular matrix channel or the cell chamber through the separation space formed in the first station-micro-dam structure.

[0021] In addition, the bio-tissue chip may further include a cell inlet and a residue outlet communicating through the cell chamber; and a second station-micro-weir structure having a lower end spaced from the floor, wherein the second station-micro-weir structure may be formed adjacent to the cell chamber and the residue outlet.

[0022] At this time, the first reverse micro-fine weir structure may have a rectangular cross-section, and the second reverse micro-fine weir structure may have an arc-shaped cross-section.

[0023] In addition, the bio-tissue chip may further include a culture medium supply chamber and a culture medium recovery chamber communicated through the culture medium channel; and a hydrogel inlet and a hydrogel outlet communicated through the extracellular matrix channel.

[0024] Meanwhile, the present invention provides a drug efficacy and toxicity evaluation system including a bio-tissue chip that mimics the pulmonary bronchial microenvironment.

[0025] The bio-tissue chip according to the present invention as described above has the effect of enabling easy co-culturing of lung epithelial cells and lung fibroblasts and simulating the complex environment surrounding lung epithelial cells, thereby enabling the implementation of a microenvironment of human lung bronchioles.

[0026] In addition, the biotissue chip according to the present invention has the effect of more accurately deriving toxicity results according to chemical substance treatment by implementing the microenvironment of human lung bronchioles.

[0027] In addition, by using the biotissue chip according to the present invention as an alternative testing method to animal testing, not only can the cost and time required for screening chemical toxicants be drastically reduced, but it can also be applied to various lung microenvironment studies and other organ chip studies.

[0028] The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0029] Figure 1 is a three-dimensional diagram of a bio-tissue chip mimicking the pulmonary bronchial microenvironment of the present invention.

[0030] Figure 2 is a plan view of a bio-tissue chip mimicking the pulmonary bronchial microenvironment of the present invention.

[0031] Figure 3 is a partial enlarged view of area A of Figure 2.

[0032] Figure 4 is a partially enlarged stereoscopic view of area A of Figure 2.

[0033] FIG. 5 is a three-dimensional view of a pulmonary bronchial microenvironment-mimicking bio-tissue chip according to one embodiment of the present invention, a microstructure diagram including a mixed region (B), and a schematic diagram of a state in which pulmonary epithelial cells and pulmonary fibroblasts are cultured.

[0034] Figure 6 shows (A) an actual photograph of a bio-tissue chip imitating a pulmonary bronchial microenvironment, (B) a photograph showing the result of injecting food coloring into the bio-tissue chip, and (C) an enlarged photograph showing the result of culturing lung epithelial cells and lung fibroblasts.

[0035] Figure 7 is a microscopic photograph comparing the degree of damage to lung epithelial cells after cyclohexanol treatment on a lung bronchial microenvironment-mimicking bio-tissue chip cultured with only lung epithelial cells and a lung bronchial microenvironment-mimicking bio-tissue chip co-cultured with lung epithelial cells and lung fibroblasts.

[0036] Figure 8 is a microscopic photograph comparing the degree of damage to lung epithelial cells in a bio-tissue chip simulating a lung bronchial microenvironment in which lung epithelial cells and lung fibroblasts are co-cultured with each other after treatment with cyclohexanol compared to the control group.

[0037] Figure 9 is a graph showing the results of analyzing the cytotoxicity of lung epithelial cells after treating a bio-tissue chip simulating a lung bronchial microenvironment in which lung epithelial cells and lung fibroblasts are co-cultured with cyclohexanol at various concentrations.

[0038] 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 together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided only 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. Accordingly, in some embodiments, well-known process steps, well-known device structures, and well-known techniques are not specifically described to avoid ambiguity in the interpretation of the present invention. Like reference numerals refer to like elements throughout the specification.

[0039] The terminology used herein is for the purpose of describing embodiments only and is not intended to limit the present invention. In this specification, the singular also includes the plural unless the context clearly dictates otherwise. As used herein, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components, steps, operations, and / or elements.

[0040] The identifiers used in each step are for convenience of explanation and do not indicate the order of the steps. The steps may be performed in a different order than stated, unless the context clearly dictates otherwise. In other words, the steps may be performed in the same order as stated, substantially simultaneously, or in the opposite order.

[0041] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in their common sense to those of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0042]

[0043] FIG. 1 is a three-dimensional view of a pulmonary bronchial microenvironment-mimicking bio-tissue chip, FIG. 2 is a plan view of a pulmonary bronchial microenvironment-mimicking bio-tissue chip, FIG. 3 is a partial enlarged view of area A of FIG. 2, FIG. 4 is a partial enlarged three-dimensional view of area A of FIG. 2, and FIG. 5 is a three-dimensional view of a pulmonary bronchial microenvironment-mimicking bio-tissue chip according to one embodiment of the present invention, a microstructure diagram including a mixed area (B), and a schematic diagram of a state in which pulmonary epithelial cells and pulmonary fibroblasts are cultured.

[0044] Referring to FIGS. 1 to 5, the pulmonary bronchial microenvironment-mimicking bio-tissue chip (1) that supplies a culture medium to fixed pulmonary epithelial cells of the present invention and cultures them includes a culture medium supply unit (10), a hydrogel supply unit (20), and a cell culture unit (30).

[0045] The material of the overall structure of the pulmonary bronchial microenvironment-mimicking bio-tissue chip (1) of the present invention is not particularly limited, as long as it does not inhibit or adversely affect the fixation and culture of cells. As an example, it may be polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polydioxanone (PDO), etc., and as another example, it may be polydimethylsiloxane (PDMS), etc., which is optically transparent, durable, biocompatible, and flexible.

[0046] In addition, the pulmonary bronchial microenvironment-mimicking bio-tissue chip (1) according to one embodiment of the present invention is in the form of a structure provided on a slide glass (2), and has a cell culture section (30) as the center, and a culture medium supply section (10) and a hydrogel supply section (20) arranged on the upper and lower sides, respectively. However, the pulmonary bronchial microenvironment-mimicking bio-tissue chip (1) can variously modify the arrangement of the culture medium supply section (10) or the hydrogel supply section (20) centered on the cell culture section (30) while maintaining the detailed structure of the mixing region (B) in which culture media can mutually exchange. A detailed description of the detailed structure of the mixing region (B) will be described later.

[0047] The culture solution supply unit (10) may include a culture solution channel (11a, 11b), a culture solution supply chamber (12a, 12b), and a culture solution recovery chamber (13a, 13b), and the culture solution supply chamber (12a, 12b) and the culture solution recovery chamber (13a, 13b) may be connected through the culture solution channel (11a, 11b).

[0048] The culture medium supply chamber (12a, 12b) stores a culture medium for cell culture to be supplied to the cell chamber (31), and supplies the stored culture medium to the extracellular matrix channel (21a, 21b) and the cell chamber (31) through the culture medium channel (11a, 11b).

[0049] The culture medium recovery chamber (13a, 13b) is a portion where the culture medium supplied from the culture medium supply chamber (12a, 12b) passes through the culture medium channel (11a, 11b) and is finally stored. It performs the role of recovering the culture medium contaminated after cell culture, and is used for the purpose of preventing contamination of the surrounding environment due to the culture medium contaminated with various metabolites and waste products secreted from cells after cell culture.

[0050] The culture medium channel (11a, 11b) serves as a main flow path for the culture medium that circulates from the culture medium supply chamber (22a, 22b) to the culture medium recovery chamber (23a, 23b), and supplies the culture medium to each cell fixed and cultured in the cell chamber (31) or the cell chamber (31) and the extracellular matrix channel (22a, 22b) and recovers the culture medium used for cell culture.

[0051] That is, the culture channel (11a, 11b) in the pulmonary bronchial microenvironment-mimicking bio-tissue chip (1) can be interpreted as a configuration that mimics capillaries for supplying nutrients to cells existing in a living body, and vascular endothelial cells can be further cultured inside the culture channel (11a, 11b) to mimic a form more similar to the living body environment.

[0052] The hydrogel supply unit (20) may include an extracellular matrix channel (21a, 21b), a hydrogel inlet (22a, 22b), and a hydrogel outlet (23a, 23b), and the hydrogel inlet (22a, 22b) and the hydrogel outlet (23a, 23b) may be communicated through the extracellular matrix channel (21a, 21b).

[0053] The hydrogel inlets (22a, 22b) serve to introduce hydrogel for supply to the extracellular matrix channels (21a, 21b). To this end, the hydrogel inlets (22a, 22b) may be formed to have a structure to which a syringe nozzle can be connected, but any structure capable of introducing hydrogel is not particularly limited.

[0054] The hydrogel outlet (23a, 23b) is a site where the hydrogel supplied from the hydrogel inlet (22a, 22b) passes through the extracellular matrix channel (21a, 21b) and is ultimately stored, and serves to recover hydrogel residue that is not stored or fixed in the extracellular matrix channel (21a, 21b).

[0055] The extracellular matrix channels (21a, 21b) can store and fix the hydrogel supplied from the hydrogel inlet (22a, 22b). The hydrogel formed inside the extracellular matrix channels (21a, 21b) can pass the culture medium supplied from the culture medium channels (11a, 11b) due to its inherent porous properties, and can also pass various metabolic products and waste products generated from the cell chamber (31).

[0056] More specifically, in the pulmonary bronchiole microenvironment-mimicking bio-tissue chip of the present invention, the culture medium flowing in from the culture medium channel (11a, 11b) can pass through the extracellular matrix channel (21a, 21b) and be supplied to the cell chamber (31). At this time, the flow rate of the culture medium can be reduced in the process of passing through the extracellular matrix channel (21a, 21b) from the culture medium channel (11a, 11b) and being supplied to the cell chamber (31) due to the hydrogel formed inside the extracellular matrix channel (21a, 21b), thereby allowing the pulmonary bronchiole microenvironment to be more similarly simulated.

[0057] In addition, in order to more closely mimic the pulmonary bronchiole microenvironment, pulmonary fibroblasts may be further cultured inside the extracellular matrix channels (21a, 21b). As a method for culturing pulmonary fibroblasts inside the extracellular matrix channels (21a, 21b), for example, a mixture of hydrogel and pulmonary fibroblasts may be introduced into the hydrogel inlet (22a, 22b) so that pulmonary fibroblasts may be cultured inside the extracellular matrix channels (21a, 21b). At this time, the hydrogel is mixed with the pulmonary fibroblasts to assist the three-dimensional culture of the pulmonary fibroblasts and provide fluidity to the pulmonary fibroblasts so that the pulmonary fibroblasts can move from the hydrogel inlet (22a, 22b) to the hydrogel outlet (23a, 23b). Meanwhile, as another example, a mixture of lung fibroblasts and any one of collagen hydrogel, fibronectin hydrogel, or mucin hydrogel can be introduced, thereby culturing lung fibroblasts inside the extracellular matrix channels (21a, 21b).

[0058] The cell culture unit (30) may include a cell chamber (31) in which lung epithelial cells are fixed, a cell inlet (32), and a residue outlet (33), and the cell inlet (32) and the residue outlet (33) may be communicated through the cell chamber (31).

[0059] The cell inlet (32) serves to introduce a mixture of lung epithelial cells to be supplied to the cell chamber (31). To this end, the cell inlet (32) may be formed to have a structure to which a nozzle of a syringe can be connected, but is not particularly limited as long as it has a structure capable of introducing the mixture of lung epithelial cells.

[0060] At this time, the lung epithelial cell mixture may include lung epithelial cells and an auxiliary component that assists in the circulation and fixation of lung epithelial cells. The auxiliary component mimics the extracellular matrix (ECM) existing in the microenvironment around lung epithelial cells, is essential for the three-dimensional culture of lung epithelial cells, and can play a role in maintaining the structure and function of the cultured lung epithelial cells to be similar to those in a living body. The auxiliary component is not particularly limited thereto as long as it can assist in the inflow and fixation of lung epithelial cells, and as an example, a hydrogel may be used, and as another example, any one of collagen hydrogel, fibronectin hydrogel, or elastin hydrogel may be used. Meanwhile, the hydrogel is mixed with lung epithelial cells to assist in the three-dimensional culture of lung epithelial cells, and additionally provides fluidity to the lung epithelial cells so that the lung epithelial cells can move from the cell inlet (32) to the cell chamber (31). In addition, since the hydrogel can be hardened under various conditions, when the lung epithelial cell mixture moves to the cell chamber (31) and the hydrogel contained therein hardens, the lung epithelial cells contained within the hardened hydrogel can be naturally fixed. In addition, due to the inherent porous characteristics of the hydrogel, the culture medium supplied through the extracellular matrix channels (21a, 21b) of the hydrogel can be delivered to the lung epithelial cells, thereby assisting the culture of the fixed lung epithelial cells.

[0061] The cell chamber (31) can not only store and fix the lung epithelial cell mixture supplied through the cell inlet (32), but also serve as a culture vessel for culturing the fixed lung epithelial cells. Meanwhile, the lung epithelial cells in the present invention refer to any one of the epithelial cells of the bronchi, bronchioles, or alveoli.

[0062] The residue outlet (33) serves to discharge to the outside contaminants such as metabolic products or wastes generated from lung epithelial cells while culturing lung epithelial cells fixed to the cell chamber (31), and is not particularly limited as long as it is configured in a form that can be combined with a nozzle of a tube or syringe. However, the role of discharging contaminants is not limited thereto, and not only the residue outlet (33) but also the cell inlet (32) can perform the role of dischargeing contaminants, and when the role of introducing the lung epithelial cell mixture into the cell chamber (31) is finished, the role of discharging to the outside contaminants generated while culturing lung epithelial cells fixed to the cell chamber (31) can also be performed.

[0063] In one embodiment of the present invention, the pulmonary bronchial microenvironment-mimicking bio-tissue chip may further include a reverse-micro-weave structure (40).

[0064] The reverse-micro-dam structure (40) refers to a partial blocking member, and its upper end is provided to be connected to the upper end of the adjacent part and its lower end is provided with a space separated from the bottom of the adjacent part, so that cells or hardened cells cannot pass through the space separated at the lower end, but liquid components can pass through.

[0065] The shape of the reverse-micro-weir structure (40) is not particularly limited as long as it exhibits the functionality described above, but it is preferably a micro-barrier structure having a rectangular or arc-shaped cross-section so that only the culture medium can pass through without the lung epithelial cells or lung fibroblasts escaping out of the cell chamber (31) or the extracellular matrix channel (21a, 21b). In addition, the first reverse-micro-weir structure (41) having a rectangular cross-section and the second reverse-micro-weir structure (42) having an arc-shaped cross-section, which will be described later, may be formed to have different widths.

[0066] Referring again to FIGS. 3 and 4, the pulmonary bronchial microenvironment-mimicking bio-tissue chip (1) of the present invention includes a mixing region (B) in which culture media can interact. In this case, the mixing region (B) can be interpreted as a component that mimics the pulmonary bronchial microenvironment existing in a living body.

[0067] In the mixing region (B), the culture medium channels (11a, 11b), the extracellular matrix channels (21a, 21b) and the cell chamber (31) can be arranged longitudinally in parallel, and in order to more closely mimic the pulmonary bronchiole microenvironment, the width of the cell chamber (31) is preferably wider than the width of the culture medium channels (11a, 11b), and the width of the culture medium channels (11a, 11b) is preferably wider than the width of the extracellular matrix channels (21a, 21b). In addition, it is more preferable that the culture medium channels (11a, 11b), the extracellular matrix channels (21a, 21b) and the cell chamber (31) are arranged in the order of top to bottom or bottom to top.

[0068] It is preferable that the reverse-micro-weir structure (40) be arranged in multiples in terms of efficient culture of lung epithelial cells and lung fibroblasts. At this time, the reverse-micro-weir structure (40) can be divided into a first reverse-micro-weir structure (41) having a straight cross-section and a second reverse-micro-weir structure (42) having an arc-shaped cross-section, and the first reverse-micro-weir structure (41) and the second reverse-micro-weir structure (42) can be formed so that the lower end has a space separated from the floor as described above.

[0069] It is preferable that the first station-micro-weir structure (41) is formed in the adjacent portion between the culture medium channel (11a, 11b) and the extracellular matrix channel (21a, 21b) and the adjacent portion between the extracellular matrix channel (21a, 21b) and the cell chamber (31), so that the culture medium of the culture medium channel (11a, 11b) can move to the extracellular matrix channel (21a, 21b) or the cell chamber (31) through the separation space formed in the first station-micro-weir structure (41). Meanwhile, it is preferable that the second station-micro-weir structure (42) is formed in the adjacent portion between the cell chamber (31) and the residue outlet (33).

[0070] The height of the spaced apart at the bottom of the first-station-micro-weir structure (41) and the second-station-micro-weir structure (42) is not particularly limited as long as the passage of pulmonary epithelial cells or pulmonary fibroblasts is not permitted, but may be, for example, 5 µm to 20 µm, for another example, 7 µm to 15 µm, and for yet another example, 10 µm. When the height of the spaced apart is less than 5 µm, liquid components cannot pass between the bottom of the first-station-micro-weir structure (41) and the second-station-micro-weir structure (42) and the bottom of the junction due to surface tension, and when the height of the spaced apart is greater than 20 µm, pulmonary epithelial cells or pulmonary fibroblasts can move through the spaced apart.

[0071] Meanwhile, a drug efficacy and toxicity evaluation system including a biochip (1) that mimics the pulmonary bronchial microenvironment can be constructed, and the system can evaluate drug efficacy and toxicity by measuring changes in pulmonary epithelial cells due to the injected drug.

[0072] At this time, changes in lung epithelial cells may be selected from a group consisting of damage to cultured lung epithelial cells, growth, changes in secretory components, changes in gene expression levels, changes in protein levels, and combinations thereof, but are not limited thereto, and various factors for changes in lung epithelial cells may be added.

[0073]

[0074] Hereinafter, examples will be examined to aid understanding of the present invention. However, these are merely the most preferred embodiments of the present invention and do not fully represent the technical scope of the present invention. Therefore, it should be understood that various equivalents and modifications may be substituted for these embodiments at the time of filing of the present invention.

[0075]

[0076] Example 1: Fabrication of a biotissue chip mimicking the pulmonary bronchial microenvironment.

[0077] The pulmonary bronchial microenvironment-mimicking bio-tissue chip (hereinafter referred to as the 'bio-tissue chip') of the present invention was manufactured using PDMS (polydimethylsiloxane) elastomer and slide glass.

[0078] PDMS silicone base elastomer and curing agent (SYLGARD 184 silicone elastomer kit, Dow Corning) were mixed in a ratio of 10:1 on the mold on which photoresist patterning was completed, and air bubbles generated during mixing were removed using the vacuum system of a plasma processing system (CUTE, FEMTO Science). Afterwards, the mixture was poured onto the SU-8 mold and heat-treated on a hot plate at 80°C for 10 hours to solidify. After separating it from the mold, it was cut according to the appropriate size of the chip, and then a cylindrical 1.2 mm and 8 mm diameter biopsy punch was used to punch out the culture medium supply section (culture medium channel, culture medium supply chamber, culture medium recovery chamber), hydrogel supply section (extracellular matrix channel, hydrogel inlet, hydrogel outlet) and cell culture section (cell chamber, cell inlet, residue outlet) according to their respective sizes to fabricate a PDMS-based microfluidic device.

[0079] PDMS-based microfluidic devices were cleaned by sonication in a sonicator containing isopropyl alcohol, and then completely dried using an air gun.

[0080] The washed PDMS-based microfluidic device and slide glass were dried in an 80°C dry oven for 10 minutes and then sterilized by exposure to ultraviolet light for 30 minutes. Finally, the PDMS-based microfluidic device and slide glass were placed in a plasma processor and bonded through oxygen plasma treatment to produce a biotissue chip. The fabricated biotissue chip was placed in a 35 mm petri dish and heated on a hot plate heated to 80°C for 30 minutes to strengthen the adhesion caused by the plasma treatment.

[0081] The overall size of the biotissue chip fabricated as above is 2.1 cm in width and 2.7 cm in length, and the height of all channel microstructures in the biotissue chip was 50 μm, and the height excluding the reverse micro-weir structure was confirmed to be 5 μm. Meanwhile, in the mixing region, the width of the cell chamber was confirmed to be 600 μm, the width of the extracellular matrix channel was confirmed to be 60 μm, and the width of the culture medium channel was confirmed to be 100 μm. The diameters of the cell inlet and residue outlet connected to the cell chamber were confirmed to be 1.5 mm, and the diameter of the culture medium channel connected to the culture medium supply chamber and the culture medium recovery chamber was confirmed to be 8 mm.

[0082] In addition, it was configured to include one cell chamber, two extracellular matrix channels, and two culture medium channels. The cell chamber was positioned between the two extracellular matrix channels, which were arranged longitudinally in parallel, and two culture medium channels were configured to be adjacent to each other outside the outer extracellular matrix channel.

[0083] In addition, the first station-micro-weir structure was designed to have a rectangular cross-section and a width of 100 μm, and was positioned at the adjacent portion between the culture medium channel and the extracellular matrix channel and the adjacent portion between the extracellular matrix channel and the cell chamber, so that the mixture of pulmonary epithelial cells and pulmonary fibroblasts accumulated without escaping to the outside.

[0084] In addition, the second station-micro-dam structure was designed to have an arc-shaped cross-section and a width of 60㎛, and was positioned adjacent to the cell chamber and the residue outlet, so that the lung epithelial cell mixture accumulated without escaping toward the residue outlet, thereby ensuring smooth injection and allowing only the pressure generated during injection to escape toward the residue outlet.

[0085]

[0086] Example 2: Fabrication of a biotissue chip cultured with lung bronchiole epithelial cells

[0087] First, a hydrogel solution was separately injected into the extracellular matrix channel using a pipette through the hydrogel inlet of the bio-tissue chip of the present invention manufactured in Example 1. It was confirmed that the hydrogel solution formed a boundary between the culture medium channel and the extracellular matrix channel, and between the extracellular matrix channel and the cell chamber, with the first station-micro-weir structure as the boundary. In addition, it was confirmed that the hydrogel was reproducibly formed within the extracellular matrix channel.

[0088] Next, 100 ml of culture medium was loaded into each culture medium supply chamber, and the flow of culture medium was connected to each culture medium supply chamber using a pipette. Then, the culture medium was filled into each culture medium recovery chamber to ultimately create a medium flow. At this time, it was confirmed that the medium formed a boundary between the culture medium channel and the cell chamber due to surface tension with the first station-micro-weir structure as the boundary.

[0089] Next, a mixture of hydrogel solution and ATCC bronchial epithelial cells was injected into the cell chamber using a pipette into the cell inlet, and then the cells were cultured in a humidified incubator at 37°C and 5% CO2 in DMEM culture medium supplemented with P / S (100 U ml-1 penicillin / 100 U ml-1 streptomycin, Sigma-Aldrich Co., MO), 10% (v / v) FBS, and 10% NEAA.

[0090] The time required for the injection process of the lung epithelial cell mixture using the bio-tissue chip of the present invention was confirmed to be less than 5 minutes per chip, demonstrating reproducible results. This experimental time was confirmed to be significantly reduced compared to the 3 hours per chip required for the injection process of the cardiomyocyte mixture using the existing pericyte-mimicking cardiac chip.

[0091] Next, the hydrogels included in each lung epithelial cell mixture were hardened to induce three-dimensional hydrogel formation.

[0092] The culture medium was replaced with fresh culture medium daily to provide continuous medium flow and fresh nutrients, and a biotissue chip containing cultured lung epithelial cells was produced by culturing the cells in a humidified incubator at 37°C and 5% CO2 for 5 days using the above culture method.

[0093]

[0094] Example 3: Fabrication of a biotissue chip co-cultured with lung bronchiole epithelial cells and lung fibroblasts.

[0095] First, a mixture of hydrogel solution and ATCC lung fibroblasts (MRC-5) was separately injected into the extracellular matrix channel using a pipette through the hydrogel inlet of the bio-tissue chip manufactured in Example 1. It was confirmed that the hydrogel solution formed a boundary between the culture medium channel and the extracellular matrix channel, and between the extracellular matrix channel and the cell chamber, with the first station-micro-weir structure as the boundary. In addition, it was confirmed that the hydrogel was reproducibly formed within the extracellular matrix channel.

[0096] Next, 100 ml of culture medium was loaded into each culture medium supply chamber, and the flow of culture medium was connected to each culture medium supply chamber using a pipette. Then, the culture medium was filled into each culture medium recovery chamber to ultimately create a medium flow. At this time, it was confirmed that the medium formed a boundary between the culture medium channel and the cell chamber due to surface tension with the first station-micro-weir structure as the boundary.

[0097] Next, a mixture of hydrogel solution and ATCC bronchial epithelial cells was injected into the cell chamber using a pipette into the cell inlet, and then the cells were cultured in a humidified incubator at 37°C and 5% CO2 in DMEM culture medium supplemented with P / S (100 U ml-1 penicillin / 100 U ml-1 streptomycin, Sigma-Aldrich Co., MO), 10% (v / v) FBS, and 10% NEAA.

[0098] The time required for the injection process of the lung epithelial cell mixture using the bio-tissue chip of the present invention was confirmed to be less than 5 minutes per chip, demonstrating reproducible results. This experimental time was confirmed to be significantly reduced compared to the 3 hours per chip required for the injection process of the cardiomyocyte mixture using the existing pericyte-mimicking cardiac chip.

[0099] Next, the hydrogels included in the mixture of lung fibroblasts and lung epithelial cells were cured to induce three-dimensional hydrogel formation.

[0100] The culture medium was replaced with fresh culture medium daily to provide continuous medium flow and fresh nutrients, and a biotissue chip co-cultured with lung epithelial cells and lung fibroblasts was produced by culturing the cells in a humidified incubator at 37°C and 5% CO2 for 5 days using the above culture method.

[0101] Referring to Fig. 6(c), it can be confirmed that lung epithelial cells and lung fibroblasts are well cultured in the cell chamber and extracellular matrix channel of the biotissue chip, respectively.

[0102]

[0103] Experimental Example 1: Functional Evaluation of the Biotissue Chip of Example 1

[0104] It was evaluated whether the biotissue chip manufactured in Example 1 functions normally.

[0105] After setting the red, blue, and yellow food colorings to appropriate concentrations and viscosities, 100 ml of the red food coloring solution was loaded into each of a set of culture solution supply chambers and a culture solution recovery chamber, and the food coloring solution was filled into the inside of the culture solution channel using a pipette, and then 100 ml was also loaded into the remaining set of culture solution supply chambers and culture solution recovery chambers to connect the flow of the red food coloring solution between each chamber.

[0106] Afterwards, the blue food coloring solution was injected into each extracellular matrix channel at a rate of 20 ml / min using a syringe pump (NE-1000, NEWERA), and the yellow food coloring solution was similarly injected into the cell chamber at a rate of 20 ml / min using a syringe pump (NE-1000, NEWERA).

[0107] Referring to Fig. 6 (a) and (b), it was confirmed that each food coloring solution formed a boundary between the culture medium channel and the extracellular matrix channel, and between the extracellular matrix channel and the cell chamber, by surface tension, with the first station-micro-weir structure as the boundary, thereby confirming that the selective liquid was successfully separated and injected.

[0108]

[0109] Experimental Example 2: Drug toxicity evaluation using the biotissue chips of Examples 2 and 3.

[0110] In order to evaluate the drug toxicity of the bio-tissue chips manufactured in Examples 2 and 3, cyclohexanol, known to be toxic to the lungs, was treated at a concentration of 100 μM in each culture solution supply chamber of the bio-tissue chips manufactured in Examples 2 and 3, and changes after 24 hours were observed using an optical microscope.

[0111] Referring to Fig. 7, it was confirmed that both the bio-tissue chips of Examples 2 and 3 killed lung epithelial cells due to the pulmonary toxicity of cyclohexanol, and in particular, in the case of the bio-tissue chip of Example 3, it was confirmed that the degree of cell death was reduced due to the presence of lung fibroblasts cultured in the extracellular matrix channel. This means that cyclohexanol can start from the culture medium channel, pass through the extracellular matrix channel, and then reach the lung epithelial cells cultured in the cell chamber, and it was confirmed that when lung fibroblasts are cultured in the extracellular matrix channel, the microenvironment of the lung bronchiole can be more similarly simulated.

[0112]

[0113] Experimental Example 3: Drug toxicity evaluation using the biotissue chip of Example 3 and the control group.

[0114] In order to evaluate the drug toxicity of the bio-tissue chip manufactured in Example 3, cyclohexanol, known to be toxic to the lungs, was treated at a concentration of 100 μM in each culture medium supply chamber of the bio-tissue chip manufactured in Example 3, and changes after 24 hours were observed using an optical microscope, and the degree of damage to lung epithelial cells was compared and analyzed with the control group. At this time, a medium not treated with cyclohexanol was used as the control group in the bio-tissue chip manufactured in Example 3.

[0115] Referring to Fig. 8, it can be confirmed that the control group was cultured normally without death of lung epithelial cells, but in the case of the bio-tissue chip of Example 3 treated with cyclohexanol, it was confirmed that most of the lung epithelial cells were killed due to the lung toxicity of cyclohexanol. This means that cyclohexanol can start from the culture medium channel, pass through the extracellular matrix channel, and then reach the lung epithelial cells cultured in the cell chamber, and thus it was further confirmed that the bio-tissue chip of the present invention can be a low-cost, rapid, and accurate drug toxicity evaluation platform that can replace animal testing.

[0116]

[0117] Experimental Example 4: Evaluation of drug concentration-dependent toxicity in lung epithelial cells using the biotissue chip of Example 3.

[0118] In order to evaluate the toxicity of lung epithelial cells according to drug concentration using the biotissue chip manufactured in Example 3, cyclohexanol, known to be toxic to lungs, was treated at concentrations of 0 μM, 10 μM, and 100 μM to each culture medium supply chamber of the biotissue chip manufactured in Example 3, and the cytotoxicity of lung epithelial cells was analyzed.

[0119] Referring to Fig. 9, it was confirmed that the toxicity of lung epithelial cells increased as the concentration of cyclohexanol increased, and in particular, when treated with a concentration of 100 μM, the cytotoxicity of lung epithelial cells increased rapidly, unlike when treated with a concentration of 10 μM. This means that the concentration of cyclohexanol can be normally reflected in lung epithelial cells, and thus, it was further confirmed that the bio-tissue chip of the present invention can serve as a low-cost, rapid, and accurate drug toxicity evaluation platform that can replace animal testing.

[0120] The embodiments of the present invention described above are merely exemplary, and the scope of protection of the present invention may include various modifications and equivalent examples thereof by a person having ordinary skill in the art of the present invention.

[0121] 1: A biotissue chip that mimics the pulmonary bronchial microenvironment.

[0122] 2: Slide glass

[0123] 10: Culture solution supply section

[0124] 11: Culture channel

[0125] 12: Culture solution supply chamber

[0126] 13: Culture medium recovery chamber

[0127] 20: Hydrogel supply unit

[0128] 21: Extracellular matrix channel

[0129] 22: Hydrogel inlet

[0130] 23: Hydrogel outlet

[0131] 30: Cell culture section

[0132] 31: Cell chamber

[0133] 32: Cell inlet

[0134] 33: Residue outlet

[0135] 40: Reverse-micro-dam structure

[0136] 41: Station 1 - Fine-grained structure

[0137] 42: Station 2 - Fine-grained structure

Claims

1. In a bio-tissue chip simulating the microenvironment of the pulmonary bronchial tubes, cultured by supplying culture medium to fixed pulmonary epithelial cells, The above biotissue chip includes a culture medium channel, an extracellular matrix channel, and a cell chamber. The above lung epithelial cells are fixed in the cell chamber, A bio-tissue chip simulating the microenvironment of a pulmonary bronchial tube, wherein the culture medium is supplied from the culture medium channel through the extracellular matrix channel to the cell chamber.

2. In claim 1, A bio-tissue chip mimicking the microenvironment of a pulmonary bronchial tube, characterized in that the extracellular matrix channel has a hydrogel formed inside.

3. In claim 2, A bio-tissue chip simulating a pulmonary bronchial microenvironment, characterized in that the flow rate of the culture medium decreases in the process of being supplied from the culture medium channel to the cell chamber through the extracellular matrix channel.

4. In claim 2, A bio-tissue chip mimicking the microenvironment of a pulmonary bronchial tube, characterized in that the extracellular matrix channel has pulmonary fibroblasts further cultured inside.

5. In claim 2, A bio-tissue chip mimicking the microenvironment of a pulmonary bronchial tube, characterized in that the culture channel has further cultured vascular endothelial cells inside.

6. In claim 1, A pulmonary bronchial microenvironment-mimicking biotissue chip, characterized in that the above pulmonary epithelial cells are fixed in the cell chamber by being mixed with any one of a hydrogel, a collagen hydrogel, a fibronectin hydrogel, or an elastin hydrogel.

7. In claim 1, The above biotissue chip includes a mixing area where the culture medium can interact with each other, A bio-tissue chip mimicking the microenvironment of a pulmonary bronchial tube, characterized in that the culture medium channel, the extracellular matrix channel, and the cell chamber in the above mixing region are arranged in parallel in the longitudinal direction.

8. In claim 7, The width of the above cell chamber is wider than the width of the above culture medium channel, A bio-tissue chip mimicking the microenvironment of a pulmonary bronchial tube, characterized in that the width of the culture medium channel is wider than the width of the extracellular matrix channel.

9. In claim 1, The above biotissue chip further includes a first-stage micro-dam structure having a space separated from the bottom at the bottom, A bio-tissue chip simulating a pulmonary bronchial microenvironment, characterized in that the first-stage micro-dam structure is formed in the adjacent portion between the culture medium channel and the extracellular matrix channel and in the adjacent portion between the extracellular matrix channel and the cell chamber.

10. In claim 9, A bio-tissue chip simulating a pulmonary bronchial microenvironment, characterized in that the culture medium moves into the extracellular matrix channel or the cell chamber through the separation space formed in the first station-micro-dam structure.

11. In claim 9, The above bio-tissue chip further comprises a cell inlet and a residue outlet communicating through the cell chamber; and a second station-micro-dam structure having a lower end spaced from the floor, A bio-tissue chip mimicking the microenvironment of a pulmonary bronchial tube, characterized in that the second-stage micro-dam structure is formed adjacent to the cell chamber and the residue outlet.

12. In claim 11, The above first-order reverse micro-fine structure has a rectangular cross-section, A bio-tissue chip mimicking the microenvironment of a pulmonary bronchial tube, characterized in that the second-stage micro-fine meshwork structure has an arc-shaped cross-section.

13. In claim 9, A pulmonary bronchial microenvironment-mimicking bio-tissue chip, characterized in that the bio-tissue chip further includes a culture medium supply chamber and a culture medium recovery chamber communicated through the culture medium channel; and a hydrogel inlet and a hydrogel outlet communicated through the extracellular matrix channel.

14. A drug efficacy and toxicity evaluation system comprising a bio-tissue chip mimicking the pulmonary bronchial microenvironment of any one of claims 1 to 13.

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