Cell culture exposure unit, cell culture exposure device and bionic breathing type exposure system

Abstract

The utility model belongs to the field of in-vitro cell culture and exposure, and particularly relates to a cell culture exposure unit, a cell culture exposure device and a bionic breathing type exposure system. The cell culture exposure unit is provided with a microporous membrane, a gas chamber and a liquid chamber, the gas chamber and the liquid chamber are located on the two sides of the microporous membrane respectively, a refining baffle is arranged at the position, close to the microporous membrane, in the liquid chamber, and the refining baffle is provided with a microporous structure used for refining CO2 exhaled by the CO2 exhaling device. And the plate surface of the refining baffle plate faces the microporous membrane within the range of the plate surface. The device can better simulate that CO2 in the body stably and naturally passes through a gas-blood barrier, can adapt to different hierarchical positions and shunt flow of a bronchial tree, and is more objective and real in bionic simulation of inhalation exposure and in-vivo environment, thereby being beneficial to obtaining more real and scientific research results.

Description

Technical Field

[0001] This utility model belongs to the field of in vitro cell culture and exposure, specifically relating to a cell culture exposure unit, a cell culture exposure device, and a biomimetic respiratory exposure system. Background Technology

[0002] Respiration is a vital process for maintaining oxygen and CO2 balance in the body and ensuring metabolism. It involves three interconnected and simultaneous processes: pulmonary ventilation, pulmonary gas exchange, and gas exchange between blood and tissue cells. Harmful gases, particulate matter, and targeted therapies involving inhaled drug aerosols all enter the body through respiration. Establishing respiratory exposure systems and conducting respiratory exposure experiments are crucial for understanding the distribution of inhaled exogenous substances in the respiratory tract, obtaining exposure doses in the respiratory tract, and further assessing intra-exposure dose and dose-response relationships.

[0003] The inventor's research group has filed a Chinese invention patent application (publication date: August 12, 2022, application publication number: CN 114898637 A) disclosing a CO2 supply device and method for simulating CO2 release in vitro. This invention constructs a culture unit by placing an inner container with a porous membrane inside an outer container. The porous membrane is used for cell growth and divides the culture unit into gas chambers and liquid chambers. CO2 is supplied to the liquid chamber via a hydrophobic and breathable membrane device. While this device can simulate the gas-liquid interface environment of respiratory cells exposed to inhaled substances to some extent, it does not simulate the constant temperature environment of the human body. Furthermore, the CO2 introduced into the liquid chamber forms large bubbles that spray out of the liquid surface during the experiment. The resulting gas impact may cause some damage to the cells and also causes liquid to splash into the cell culture chamber, thus disrupting the gas-liquid interface state. This is inconsistent with the process by which CO2 gas is smoothly and naturally expelled from the lungs through the air-blood barrier in vivo.

[0004] The inventor's research group previously disclosed a Chinese invention patent application for an aerosol exposure system based on biomimetic breathing (application publication date: October 25, 2022, application publication number: CN 115232733 A), which includes a breathing power device (i.e., a biomimetic breathing device) and a biomimetic oral cavity, a biomimetic nasal cavity, and a bronchial tree connected to the breathing power device. The bronchial tree is connected to three biomimetic lung units, and the three biomimetic lung units are all connected to a CO2 supply device (i.e., a CO2 exhalation device) through CO2 pipelines.

[0005] The above aerosol exposure system can introduce CO2 according to the respiratory rhythm of inhalation and exhalation, thereby achieving high O2 during inhalation and high CO2 during exhalation. This better simulates the fluid dynamics experienced by respiratory cells and the gaseous environment in which the cells are located, and can evaluate the changes in pH environment caused by different CO2 concentrations and the resulting changes in cell growth status. However, it does not provide specific cell culture and exposure devices and methods adapted to different shunt flow rates of the bronchial tree, supporting cell culture, exposure, and CO2 introduction.

[0006] Overall, existing devices and methods cannot provide cell culture and exposure to different levels of the bronchial tree and shunt flow rates, nor can they provide a fine, uniform, and large-volume exhaled CO2 gas environment at the gas-liquid interface. This results in a lack of authenticity and scientific rigor in studies on the effects of different concentrations of CO2 on extracellular fluid pH and cells caused by inhalation exposure and various in vivo causes (such as excessive CO2 production, hyperventilation or ventilation disorders, and metabolic acid-base imbalance). Utility Model Content

[0007] The first objective of this invention is to provide a cell culture exposure unit that addresses the problem that existing technologies cannot simulate the smooth and natural passage of CO2 through the gas-blood barrier in vivo.

[0008] The second objective of this invention is to provide a biomimetic respiratory exposure system to solve the problem that the exposure units of existing systems cannot be adapted to different levels of the bronchial tree and the shunt flow rate.

[0009] The third objective of this invention is to provide a cell culture exposure device to further address the problems of inconvenient installation of cell culture exposure units and the inability to simulate the constant temperature environment in vivo.

[0010] To achieve the first objective mentioned above, the technical solution adopted by this utility model is as follows:

[0011] A cell culture exposure unit includes a microporous membrane for cell growth and a gas chamber and a liquid chamber located on both sides of the microporous membrane. The gas chamber is connected to a bronchial tree, and the liquid chamber is connected to a CO2 exhalation device. A refining baffle is disposed in the liquid chamber near the microporous membrane. The refining baffle has a microporous structure for refining the CO2 exhaled by the CO2 exhalation device, and the baffle surface faces the microporous membrane located within the baffle surface area.

[0012] This invention is an improved invention. It uses a fine baffle to transform the CO2 exhaled by the CO2 exhalation device into dense, fine, and uniform CO2 bubbles, avoiding excessive force of exhaled CO2 gas that could damage cells or disrupt the gas-liquid interface. At the same time, it restores the stable and orderly CO2 gas environment in which cells exist, better simulating the process of CO2 passing through the gas-blood barrier smoothly and naturally.

[0013] Preferably, the refining baffle is composed of multiple layers of microporous material, wherein the pore size of the microporous material is ≤0.42mm, the number of layers is ≥2, and the total thickness of the microporous material is 0-4mm. Commercially available refining plates or similar materials can be selected from this type of microporous material.

[0014] Preferably, at least a portion of the wall surface of the gas chamber is made of a soft, elastic material, so that the gas chamber has an inflated state when gas is inlet and a concave state when gas is outlet.

[0015] Preferably, the cell culture exposure unit includes a culture tank and a sealing cover for sealing the culture tank. The culture tank is provided with cell chambers having the microporous membrane. The chamber between the sealing cover and the cell chambers forms the gas chamber, and the chamber between the cell chambers and the culture tank forms the liquid chamber. An air inlet pipe for introducing gas into the gas chamber is connected to the sealing cover.

[0016] More preferably, the sealing cap is made of a soft, elastic material.

[0017] More preferably, the sealing cap is made of a double-layer material, with the inner layer being a soft elastic material and the outer layer being a rigid material to restrict the shape of the inner layer after it bulges out, and the outer layer being an upwardly convex hemispherical shape.

[0018] Preferably, the sealing cap is a flat plate structure or a raised structure with the inner side bulging upward, and the raised portion forms an expansion cavity.

[0019] Preferably, the liquid chamber is connected to a CO2 exhalation conduit for exhaling CO2 into the microporous membrane, and the outlet of the CO2 exhalation conduit is a funnel-shaped opening with a gradually increasing degree.

[0020] To achieve the second objective mentioned above, the technical solution adopted by this utility model is as follows:

[0021] A biomimetic breathing exposure system includes a biomimetic breathing device, a CO2 exhalation device, and a bronchial tree. The biomimetic breathing device is connected to the inlet of the bronchial tree. It also includes at least one cell culture exposure device, which comprises the aforementioned cell culture exposure unit. At least a portion of the wall surface of the gas chamber is made of a soft, elastic material, allowing the gas chamber to have an inflated state during air intake and a concave state during air exhaust. The gas chamber is connected to a branch of the bronchial tree, and the position of the gas chamber and its volume in the inflated state match the corresponding branch end position and its shunting ratio or target shunting ratio. The liquid chamber is connected to the CO2 exhalation device.

[0022] This novel biomimetic respiratory exposure system uses a cell culture exposure unit with both inflated and deflated states. The inflated volume is adapted to the shunting ratio of the corresponding branch of the bronchial tree or to the target shunting ratio. It can reproduce the shunting situation of airflow through the bronchial tree under different lung signs, and better adapt to different levels of the bronchial tree and shunting flow rates. This allows for more realistic and scientific research on changes in the airflow distribution ratio inside the lungs caused by inhalation exposure and various causes in vivo (such as lung surgery, atelectasis, excessive CO2 production in the body, hyperventilation or ventilation impairment, and metabolic acid-base imbalance in the body), as well as the changes in extracellular fluid pH and the effects on cells caused by different concentrations of CO2.

[0023] To achieve the third objective mentioned above, the technical solution adopted by this utility model is as follows:

[0024] A cell culture exposure device includes a water bath container and the cell culture exposure unit disposed in the water bath container.

[0025] This invention places the cell culture exposure unit in a water bath container, which not only simulates the constant temperature environment of the human body, but also provides support conditions for the cell culture exposure unit. It can adapt to the spatial distribution of different levels of the bronchial tree in the XYZ directions, avoiding problems such as the inconvenience of loading and placing in different spatial positions, making the device more compatible and adaptable, and easier to operate.

[0026] Preferably, the number of cell culture exposure units is 2 to 10, and the distribution position of the cell culture exposure units and the size of their gas chambers are adapted to the position, distribution coefficient, and flow rate or target distribution coefficient and flow rate of the terminal branches of the bronchial tree. At least part of the cavity wall surface of the gas chamber of the cell culture exposure unit is made of a soft elastic material so that the gas chamber has an inflated state when gas is inhaled and a concave state when gas is exhaled; the volume of each gas chamber in the inflated state is the same or different, so as to match the corresponding branches of the same or different levels of the bronchial tree respectively. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure of the first embodiment of the cell culture exposure device of this utility model;

[0028] Figure 2 This is a schematic diagram of the second embodiment of the cell culture exposure device of this utility model;

[0029] Figure 3 This is a schematic diagram of the third embodiment of the cell culture exposure device of this utility model;

[0030] Figure 4 This is a schematic diagram of the fourth embodiment of the cell culture exposure device of this utility model;

[0031] Figure 5 This is a schematic diagram of the fifth embodiment of the cell culture exposure device of this utility model;

[0032] Figure 6 This is a top view of the sixth embodiment of the cell culture exposure device of this utility model;

[0033] Figure 7 This is a side view of the seventh embodiment of the cell culture exposure device of this utility model;

[0034] Figure 8 This is a schematic diagram of the eighth embodiment of the cell culture exposure device of this utility model;

[0035] Figure 9 This is a schematic diagram of the structure of the biomimetic breathing exposure system of this utility model;

[0036] Among them, 1-water bath container; 2-culture unit; 3-culture tank; 4-sealing cover; 5-cell chamber; 6-microporous membrane; 7-gas chamber; 8-liquid chamber; 9-refined baffle; 10-CO2 exhalation pipeline; 100-sealing structure; 11-inlet pipeline; 12-outlet pipeline; 13-gas pipeline; 14-water bath tank; 15-water bath cover; 16-connecting flange; 17-bolt; 18-liquid inlet pipe; 19-liquid outlet pipe; 20-bionic breathing device; 21-bronchial tree; 22-CO2 exhalation device; 23-water bath machine. Detailed Implementation

[0037] This biomimetic respiratory exposure system is designed to address the limitations of existing devices in providing cell culture and exposure environments that cannot adapt to different levels of the bronchial tree and shunt flow rates, nor can they provide a fine, uniform, and large-volume exhaled CO2 gas environment at the gas-liquid interface. It provides a more objective and realistic testing device and method for conducting in vitro and in vivo studies on the effects of different concentrations of CO2 on extracellular fluid pH and cells caused by various factors (such as excessive CO2 production, hyperventilation or ventilatory obstruction, and metabolic acid-base imbalance).

[0038] The technical concept of this utility model is to construct a closed, constant-temperature cell culture exposure device that is well adapted to the stratification and diversion of the bronchial tree and has a better biomimetic gas-liquid interface, based on the actual bronchial tree and the in vivo environment of the cells, so as to further improve the authenticity and objectivity of the biomimetic respiratory cell culture exposure experiment.

[0039] Furthermore, by optimizing the number, location, material, and air intake and exhaust channels of the culture units, the biomimicry of the device can be further improved, thereby enhancing the accuracy of the results of cell culture exposure experiments (where culture serves as the control group and exposure serves as the treatment group, depending on the experimental objective).

[0040] The implementation process of this utility model will be described in detail below with reference to specific embodiments.

[0041] I. Specific Embodiments of the Cell Culture Exposure Unit and Cell Culture Exposure Device of this Utility Model

[0042] Example 1

[0043] The cell culture exposure device in this embodiment, such as Figure 1 As shown, it includes a water bath container 1 and a culture unit 2 disposed in the water bath container 1.

[0044] Culture unit 2, a key component of the entire device, enables cell respiration and exposure under biomimetic respiration conditions. It simulates the environment of lung cells, connecting to the branch ends of the bronchial tree to receive incoming air while simultaneously contacting a liquid environment, mimicking the gas-liquid interface. Specifically, Figure 1 In the culture unit 2, there are culture tank 3 and sealing cover 4 of the sealed culture tank 3. Cell chambers 5 are provided in the culture tank 3. The bottom wall of the cell chamber 5 is a microporous membrane 6 for cell adhesion and growth.

[0045] The chamber between cell chamber 5 and the sealing cap 4 constitutes the gas chamber 7, and the chamber between cell chamber 5 and the culture tank 3 constitutes the liquid chamber 8. Gas chamber 7 receives incoming air (corresponding to inhaled air during respiration), which is absorbed and metabolized by the cells growing on the microporous membrane 6 of cell chamber 5. The culture medium contained in liquid chamber 8 comes into contact with the cells growing on the microporous membrane 6 of cell chamber 5. Simulated exhaled CO2 in liquid chamber 8 flows through the cells and enters gas chamber 7, mixing with the existing gas in gas chamber 7 before flowing out, thus completing one simulated respiration process. Gas chamber 7 and liquid chamber 8 are distinguished by the state of the cells simulating respiration at the gas-liquid interface. The fact that liquid cannot enter one side of gas chamber 7 does not mean that liquid can not enter; during adherent cell culture, a portion of the culture medium is allowed to achieve cell adhesion and growth.

[0046] The culture tank 3 has an opening at the top, and a sealing groove is formed around the perimeter of the upper plane of the culture tank 3. A sealing ring corresponding to the sealing groove is provided on the sealing cover 4. After the sealing cover 4 and the culture tank 3 are closed, the gas chamber 7 and liquid chamber 8 in the culture unit 2 are isolated from external air. There are no special requirements for the shape of the culture tank 3; for example, it can be cylindrical. Figure 2 ), hemispherical ( Figure 7 ) or irregular shapes, such as combinations of curved cylindrical segments and cylindrical segments ( Figure 3 , Figure 4 ).

[0047] Cell chambers 5 are set within the culture tank 3. Each cell chamber 5 is open and concave, similar to commercially available Transwell chambers. Cell chambers 5 can be suspended or placed within the culture tank 3. A refining baffle 9 is positioned below the microporous membrane 6 of each cell chamber 5 within the culture tank 3. The refining baffle 9 refines the CO2 exhaled from the liquid chamber 8 of the culture tank 3 towards the microporous membrane 6, producing uniform, dense, and fine bubbles. This prevents damage to the cells growing on the microporous membrane 6 and more objectively and realistically simulates the smooth and natural crossing of the air-blood barrier by exhaled gases. To ensure uniform action of exhaled gases on the cells growing on the microporous membrane 6, both the refining baffle 9 and the microporous membrane 6 are arranged horizontally. The microporous membrane 6 is located within the surface area of ​​the refining baffle 9. The refining baffle 9 may or may not contact the microporous membrane 6. The refining baffle 9 is made of a microporous material and refines, homogenizes, and densifies the exhaled CO2 through its own microporous structure. The thickness of the baffle 9 can be 0-4mm, the micropore diameter can be ≤0.42mm, or a porous material with micropore or nanopore diameter can be used; to simplify the arrangement, multiple layers of porous material with micropore diameter ≤0.42mm can also be stacked to achieve the above-mentioned purpose of refining, homogenizing and densifying.

[0048] The culture tank 3, liquid chamber 8, is also connected to a CO2 exhalation pipe 10. The CO2 exhalation pipe 10 is connected to a CO2 exhalation device, which exhales a certain concentration of CO2 according to respiratory rhythm requirements or experimental requirements. This CO2 then enters the culture medium in the liquid chamber 8 through the outlet of the CO2 exhalation pipe 10. The outlet of the CO2 exhalation pipe 10 faces the refinement baffle 9. The outlet of the CO2 exhalation pipe 10 is a funnel-shaped opening with a gradually increasing angle. In other implementations, this outlet can also be a shower head structure, a conical opening, or an outlet of equal diameter with the same outer diameter as the CO2 exhalation pipe 10. Figure 4 As shown, when using the equal-diameter air outlet, the air outlet does not need to extend into the liquid chamber 8; it can be connected to the corresponding CO2 exhalation outlet on the bottom wall of the culture tank 3.

[0049] There are no special requirements for the shape of the CO2 exhalation tubing 10; it can be... Figure 1 , Figure 2 The U-shape in the middle facilitates gas flow and can also provide Figure 3 , Figure 4 The zigzag shape in the design reduces the volume occupied by the pipe. In addition, a sealing structure 100 is provided at the air inlet end of the CO2 exhalation pipe 10 to control the opening and closing of the CO2 exhalation pipe 10 as needed.

[0050] The sealing cap 4 is adapted to the culture tank 3 and is used to seal the culture tank 3. Figure 1 In the middle, the sealing cap 4 is cylindrical in shape, with its inner side bulging upward to form a hemispherical expansion cavity. This hemispherical expansion cavity expands the space of the gas chamber 7, serving as a gas buffer and helping the gas to mix before exhalation. In other embodiments, such as Figure 3 , Figure 4 As shown, the sealing cap 4 can also be a hemispherical shell, with a hemispherical expansion cavity formed inside the hemispherical shell. Of course, the sealing cap 4 can also be flat, mainly relying on the space of the cell chamber 5 itself as the gas chamber 7.

[0051] The sealing cap 4 can be made of a soft, elastic material or a rigid, inelastic material. Rigid, inelastic materials include glass, plastic, and metal, while elastic materials include rubber and latex. When a soft, elastic material is used, it exhibits an inflated state during air intake and a concave state during air exhaust. Furthermore, its inflated volume can be designed to adapt to the branching ratios of different levels of the bronchial tree, thereby simulating real-world pressure and fluid dynamic changes within the lungs. Additionally, when the sealing cap 4 is flat and made of a non-elastic material, such as… Figure 2 As shown, in order to buffer the internal pressure, the sealing cover 4 is formed by the upper cover, the lower cover and the side cover, which together form a cover with a central cavity. The vent pipe 12 connects to the central cavity. The lower cover has circumferential holes distributed along the center line of the gas chamber 7, so that the gas chamber 7 is connected to the central cavity of the sealing cover 4 and then discharged through the vent pipe.

[0052] Figure 1 In the middle, the sealing cover 4 is connected to an air inlet pipe 11 and an air outlet pipe 12. The air inlet pipe 11 and the air outlet pipe 12 are respectively connected to the corresponding interfaces of the biomimetic breathing device to simulate breathing intake and exhaust. In order to facilitate the uniform dispersion of intake air onto the cells growing on the microporous membrane 6, the air inlet pipe 11 extends into the gas chamber 7 and is close to the microporous membrane 6. The outlet of the air inlet pipe 11 is a flared structure with a gradually increasing opening. Figure 1 Specifically, it can be trumpet-shaped, but also conical, showerhead-shaped, etc. The inlet of the air outlet pipe 12 is far from the microporous membrane 6 to better simulate exhaled gas. An opening can be made on the side of the sealing cover 4 near the gas chamber 7, through which the air outlet pipe 12 passes through the sealing cover 4. Alternatively, it can be as follows... Figure 2 As shown, after the gas outlet pipe 12 is connected to the central cavity of the sealing cover 4, a hole is opened on the lower cover of the sealing cover 4 or multiple small holes are opened circumferentially along the center line of the gas chamber 7.

[0053] In other implementation scenarios, such as Figure 3 , Figure 4 As shown, only one gas pipeline 13 can be set up, which performs air intake and exhaust actions according to the breathing rhythm, making the structural setup simpler.

[0054] The water bath container 1 simulates the constant-temperature environment inside the body where the culture unit 2 is located, and also serves as the tissue body that carries the culture unit 2, simulating a more macroscopic human tissue or organ environment. The combination of the water bath container 1 and the culture unit 2 can improve the integration and biomimicry of the device, thereby restoring a more realistic and objective in vivo cell growth and exposure environment.

[0055] The water bath container 1 includes an open water bath tank 14 and a water bath cover 15 that is sealed to the water bath tank 14. The water bath tank 14 and the water bath cover 15 are provided with connecting flanges 16, and the sealing between the two is achieved by bolts 17 and sealing rings. Figure 1 In this embodiment, the sealing cap 4 of the culture unit 2 is mounted on the water bath cover 15 of the water bath container 1. When the sealing cap 4 is made of a soft, elastic material, it can be mounted on the water bath cover 15 by means of fitting, bonding, or other methods. Of course, in other implementations, the sealing cap 4 of the culture unit 2 can also be mounted separately from the water bath cover.

[0056] The water bath 14 has a square or cylindrical structure. An inlet pipe 18 and an outlet pipe 19 are located at the lower end of the water bath 14. The inlet pipe 18 is L-shaped, with its outlet located near the upper opening of the water bath 14. The outlet pipe 19 is located at the bottom of the water bath 14. Both the inlet pipe 18 and the outlet pipe 19 are connected to a water bath machine. The water bath machine generates 37°C water, which enters the water bath 14 through the inlet pipe 18, thereby simulating a constant temperature environment of 37°C inside the body.

[0057] Example 2

[0058] The cell culture exposure device in this embodiment, such as Figure 5 As shown, the device includes a water bath container 1 and two culture units 2 set in the water bath container 1. When it is necessary to combine the bronchial tree for biomimetic respiration simulation, the top wall of the gas chamber of the two culture units 2 is made of soft elastic material. The gas chamber has an inflated state when air is inhaled and a concave state when air is exhaled. The volume of each inflated chamber matches the different levels of branches of the bronchial tree to simulate the cell exposure under different bronchial tree graded flow rates.

[0059] Furthermore, unlike the structure of culture unit 2 in Example 1, culture tank 3 has a hemispherical structure, while the sealing cap 4 is made of a double-layered material. Depending on the experimental purpose, the inner and outer layers can be the same or different materials. When the inner and outer layers are the same material, they can both be rigid inelastic materials or soft elastic materials (rigid inelastic materials such as glass, plastic, and metal, and soft elastic materials such as rubber and latex). The hemispherical structure increases the gas space in the gas chamber 7, reduces the gas pressure on the cells during air intake, and simulates the spherical structure during alveolar unit expansion. When the inner and outer layers are different materials, the inner layer is a soft elastic material, and the outer layer is a rigid inelastic material. On the one hand, this increases the gas space in the gas chamber 7, reduces the gas pressure on the cells during air intake, and simulates the spherical structure during alveolar unit expansion. On the other hand, because the outer layer is inelastic, it restricts the shape of the gas chamber after gas expansion, making the pressure changes in different parts of the gas chamber 7 more uniform and stable.

[0060] Based on this embodiment, some implementations using two or more culture units 2 are as follows:

[0061] Figure 6 In this system, five culture units 2 are fixed in the same water bath container 1. The arrangement of these five culture units 2 corresponds to the five terminal positions of the bronchial tree, making it easier to connect to the corresponding bronchial levels. When the total inhalation flow rate is 500 mL, the inflated gas chambers 7 of the five culture units 2 have volumes of 70, 30, 165, 70, and 165 mL, respectively, thus accommodating different shunt flow rates.

[0062] Figure 5 This is a side view of a device equipped with two culture units 2, wherein the two culture units 2 are installed at the same height, but the gas chambers 7 are different in size.

[0063] Figure 8 In this case, the two culture units 2 are installed at different heights, and the gas chambers 7 are also different in size.

[0064] The specific embodiment of the cell culture exposure unit of this utility model is the same as the implementation of culture unit 2 in the above embodiment, and will not be described again here.

[0065] II. Specific Embodiments of the Biomimetic Breathing Exposure System of this Utility Model

[0066] Example 3

[0067] The biomimetic respiratory exposure system of this embodiment, such as Figure 9 As shown, it includes a biomimetic breathing device 20, a bronchial tree 21, a cell culture exposure device as described in the above embodiments, a CO2 exhalation device 22, and a water bath machine 23.

[0068] The biomimetic breathing device 20 is existing technology, which can simulate exhalation and inhalation in a breathing rhythm manner, and can adopt the methods in the relevant literature in the background art.

[0069] The bronchial tree 21 simulates the morphology of the bronchi in the human body. Its upstream is connected to the bionic breathing device 20, and its downstream is connected to the gas pipeline 13 of the cell culture exposure device above. Figure 9 In this implementation, the bronchial tree 21 is a single-layer hierarchical structure with two branch ends. In other implementations, the number of hierarchical layers can be more than two, and the number of branch ends can be more than five, such as ten.

[0070] The number of cell culture exposure devices is two (in other implementations, the number may be more, such as three or four), and the specific structure of the two cell culture exposure devices is the same as... Figure 4 The implementation is the same, so it will not be repeated here.

[0071] The CO2 exhalation device 22 has two CO2 exhalation outlets and is connected to the culture tank 3 through corresponding CO2 exhalation pipes 10.

[0072] The water bath 23 is connected to the inlet pipe 18 and outlet pipe 19 of the two cell culture exposure devices through corresponding connecting pipes and provides 37°C water to simulate the constant temperature environment of 37°C in vivo.

[0073] The application of the aforementioned biomimetic respiratory exposure system in in vitro exposure experiments involves selecting culture exposure units with appropriate gas chamber volumes and number distributions based on different research objectives. Specifically, it evaluates cell culture exposure based on the determination of airflow distribution ratios in each branch of the tracheal tree, CO2 ventilation flow rate and concentration that do not cause cell damage, and the damage to cells within the exposure unit caused by inhaled and exhaled gases.

[0074] 1) For the determination of the distribution ratio, the two ends of the flow sensor need to be connected to the ends of each branch of the tracheal tree, as well as the inlet and outlet ends of the culture exposure unit and the bionic alveoli (when the number of culture exposure units is less than the number of connected branches). The tracheal end of the bionic tracheal tree is connected to a bionic breathing device. A certain amount of gas is inhaled into the tracheal end of the bionic tracheal tree using the bionic breathing device, and the flow rates V1-V through each flow sensor are examined. n The branching ratio of each branch is calculated as V1 / (V1+...+V n ),...,V n / (V1+...+V n ).

[0075] 2) Determination of CO2 ventilation flow rate and concentration without causing cell damage: A biomimetic breathing device is connected to a biomimetic gas pipeline, which is connected to a culture exposure unit. The breathing tubing of the culture exposure unit is connected to a CO2 exhalation device. Different flow rates of gas are inhaled into the culture exposure unit using the CO2 exhalation device. After a certain period, cell viability is assessed using the CCK-8 assay to determine the maximum inhaled CO2 flow rate that does not cause a significant change in cell viability. Similarly, different concentrations of CO2 are inhaled into the culture exposure unit using the CO2 exhalation device. After a certain period, cell viability is assessed using the CCK-8 assay to determine the maximum inhaled CO2 concentration that does not cause a significant change in cell viability.

[0076] 3) Measurement of Cell Damage in the Exposure Unit by Inhaled / Exhaled Gases: A biomimetic breathing device is connected to a biomimetic gas duct tree, which in turn connects to the culture exposure unit. Flow sensors are connected to the ends of each branch of the gas duct tree, as well as the inlet and outlet of the culture exposure unit and the biomimetic alveoli (when the number of culture exposure units is less than the number of connected branches). The breathing tubing of the culture exposure unit is connected to a CO2 exhalation device. Gases are inhaled and exhaled into the culture exposure unit using the biomimetic breathing device, and CO2 that does not cause cell damage is exhaled into the culture exposure unit using the CO2 exhalation device. Sensors monitor the flow rate and split ratio entering the culture exposure unit. After a certain period, cell viability is assessed using the CCK-8 assay to determine the maximum total inhaled / exhaled flow rate that does not significantly change cell viability. This is the maximum usable flow rate for the culture exposure unit that does not cause cell damage. Regarding cell damage, cells are collected to measure cell viability and inflammatory factors to examine cell damage under specific inhaled / exhaled conditions.

[0077] Application Example 1

[0078] The application of the above-mentioned biomimetic respiratory exposure system in in vitro exposure experiments is explained in detail below:

[0079] 1) Apparatus Selection: Based on the bronchial tree branch 21 to be explored in the experiment, select an apparatus of appropriate size and specifications. In this embodiment, the bronchial tree branch 21 to be explored is one of the branches of the third level. When the total gas output is 500 mL, the gas output range of this branch is about 20 mL. The volume of the gas chamber after inflation can be designed to be 12-25 mL. At the same time, the volume of the simulated alveoli or culture unit connected to other branches is adjusted to meet the bronchial tree shunt ratio requirements.

[0080] 2) Constant temperature water bath of the device: Place the sterile device on the ultra-clean bench, and connect the inlet pipe 18 and outlet pipe 19 of the water bath container 1 to the outlet and inlet of the 37°C water bath machine 23, respectively, so that the 37°C water enters the water bath tank 14 in the device.

[0081] 3) Cell loading: Open the water bath cover 15 of the device, close the CO2 vent using the matching sealing structure 100, add cell culture medium to the culture tank 3 inside the device, install the cell chamber 5 onto the culture tank 3, make the microporous membrane 6 contact the liquid surface, inoculate the cell suspension into the chamber, cover the water bath cover 15, and seal the culture tank 3 with the sealing cover 4 at the same time. The water bath 14 and the surrounding area of ​​the culture tank 3 have good airtightness.

[0082] 4) Cell respiration-based adherent culture within the device: The inlet pipe 11 and outlet pipe 12 of the culture unit 2 are connected to the inlet pipe and exhalation pipe of the bronchial tree 21 of the biomimetic respiration device 20, respectively. The CO2 exhalation pipe 10 is connected to the outlet of the CO2 exhalation device 22. The respiration mode and CO2 concentration are adjusted, and the matching sealing structure 100 is opened. The cells in the culture unit 2 experience a constantly changing gas environment during inhalation and exhalation. During exhalation, dense and fine CO2 bubbles come into contact with the cells through the microporous membrane 6. During inhalation, air inhaled through the mouth and / or nose is evenly exposed to the cells in the device through the flared end of the inhalation tube 11 via the bronchial tree 21. During exhalation, CO2 is evenly diffused into the culture medium through the flared end of the CO2 exhalation tube 10. After passing through the fine baffle 9, uniform, dense, and fine CO2 bubbles are generated. After being exposed to the cells through the microporous membrane 6, they enter the gas chamber 7, mix with the inhaled gas, and enter the exhalation tube 12. Then, they enter the exhalation tube of the bronchial tree 21 and are expelled from the mouth and / or nose.

[0083] After determining the airflow distribution ratio of each branch of the tracheal tree, the CO2 ventilation flow rate and concentration that do not cause cell damage, and the parameters of cell damage caused by inhaled and exhaled gases in the exposed unit, cell respiration-based adherent culture was performed. The cell adherent culture time was 24 hours. Using cell respiration-based adherent culture can better simulate the gas-liquid interface environment of respiratory cells and more realistically reflect the physiological changes of cells under this environment.

[0084] Application Example 2

[0085] The application of the above-mentioned biomimetic respiratory exposure system in in vitro exposure experiments is explained in detail below:

[0086] Cell loading and adherence culture: Place the aseptically treated device in a laminar flow hood, open the water bath cover 15, and close the CO2 vent using the matching sealing structure 100. Add cell culture medium to the culture tank 3 inside the device. Install the cell chamber 5 onto the culture tank 3, ensuring the microporous membrane 6 is in contact with the liquid surface. Inoculate the cell suspension into the chamber, close the water bath cover 15, and simultaneously seal the culture tank 3 with the sealing cover 4. Ensure good airtightness of the water bath 14 and the periphery of the culture tank 3. Place the device in a 37℃ CO2 incubator for 24 hours to allow cell adhesion. After cell adhesion in the cell chamber 5, place the device in a laminar flow hood. Connect the inlet pipe 18 and outlet pipe 19 to the outlet and inlet of the 37℃ water bath 23, respectively, allowing 37℃ water to enter the water bath 14 of the device.

[0087] After determining the airflow distribution ratio of each branch of the tracheal tree, the CO2 ventilation flow rate and concentration that do not cause cell damage, and the parameters such as the damage of inhaled and exhaled gases to cells in the exposure unit in the manner described above, cell respiration-based adherent culture or exposure is carried out.

[0088] Cell gas-liquid interface respiration culture or exposure: After cells adhere to the wall of cell chamber 5, the culture medium is removed from cell chamber 5, leaving one side of the cell chamber as a gas chamber and the other side as a liquid chamber. Then, the cells are exposed to gas-liquid interface respiration mode according to a pre-set respiratory rhythm. Post-exposure processing: After exposure, the cells and culture medium in the exposure device can be collected, or the cells in the chamber can be further incubated or processed for subsequent experiments, depending on the purpose.

[0089] This device and exposure method can generate dense, fine, and uniform CO2 bubbles, creating a gas-liquid interface environment within cell chamber 5 that mimics actual respiration. Experiments were conducted using this method to investigate the effects of different concentrations of exhaled CO2 on the pH of the cell culture medium and cell viability. The results are shown in Tables 1 and 2. Table 3 shows the effects of different pH culture media on cells in previous experiments.

[0090] Table 1. Effects of different exhaled CO2 concentrations on pH of Beas-2b cell culture medium.

[0091]

[0092]

[0093] Table 2. Effects of different exhaled CO2 concentrations on the survival rate of Beas-2b cells.

[0094] <![CDATA[CO2 exposure concentration]]> Cell viability (%) 0 100 4％ 98.7±2.3％ 10％ 93.2±3.5％

[0095] Table 3. Effects of different pH values ​​of Beas-2b cell culture medium on the survival rate of Beas-2b cells.

[0096] pH value Cell viability (%) 3 4.02±1.07 4 4.31±1.21 5 42.51±1.15 6 92.15±3.21 7 95.41±2.56 7.21 100 8 96.68±1.80 9 88.02±5.76 10 70.34±7.07

[0097] The results above show that as the CO2 exposure concentration increases, the pH value of the Beas-2b cell culture medium continuously decreases, and the cell survival rate also decreases. This decrease in pH is due to the formation of HCO3- from CO2 during its introduction. - This is caused by the culture medium becoming weakly acidic. The decrease in cell viability is consistent with the results of previous studies using culture media with different pH values, as shown in Table 3.

Claims

1. A cell culture exposure unit, comprising a microporous membrane for cell growth and a gas chamber and a liquid chamber respectively located on both sides of the microporous membrane, wherein the gas chamber is for connection to a bronchial tree and the liquid chamber is for connection to a CO2 exhalation device, characterized in that, A refining baffle is provided in the liquid chamber near the microporous membrane. The refining baffle has a microporous structure for refining the CO2 exhaled by the CO2 exhalation device. The baffle surface faces the microporous membrane located within the baffle surface area.

2. The cell culture exposure unit as described in claim 1, characterized in that, The refined baffle is composed of multiple layers of microporous material, the pore size of which is ≤0.42mm, the number of layers is ≥2, and the total thickness of which is 0-4mm.

3. The cell culture exposure unit as described in claim 1, characterized in that, At least a portion of the wall surface of the gas chamber is made of a soft, elastic material, so that the gas chamber has an inflated state when air is introduced and a concave state when air is released.

4. The cell culture exposure unit as described in claim 1, characterized in that, The cell culture exposure unit includes a culture tank and a sealing cover for the sealed culture tank. The culture tank is provided with cell chambers having the microporous membrane. The chamber between the sealing cover and the cell chamber forms the gas chamber, and the chamber between the cell chamber and the culture tank forms the liquid chamber. An air inlet pipe for introducing gas into the gas chamber is connected to the sealing cover.

5. The cell culture exposure unit as described in claim 4, characterized in that, The sealing cap is made of a soft, elastic material.

6. The cell culture exposure unit as described in claim 4, characterized in that, The sealing cap is made of a double-layer material. The inner layer is made of a soft, elastic material, and the outer layer is made of a rigid material to restrict the shape of the inner layer after it bulges. The outer layer is a hemispherical shape that bulges upward.

7. The cell culture exposure unit as described in claim 4, characterized in that, The sealing cap is a flat plate structure or a raised structure with the inner side bulging upwards, and the raised part forms an expansion cavity.

8. The cell culture exposure unit as described in claim 1, characterized in that, The liquid chamber is connected to a CO2 exhalation tube for exhaling CO2 into the microporous membrane, and the outlet of the CO2 exhalation tube is a funnel-shaped opening with a gradually increasing degree.

9. A biomimetic breathing exposure system, comprising a biomimetic breathing device, a CO2 exhalation device, and a bronchial tree, wherein the biomimetic breathing device is connected to the inlet of the bronchial tree, characterized in that, It also includes at least one cell culture exposure device, the cell culture exposure device comprising a cell culture exposure unit as described in any one of claims 1 to 8, wherein at least a portion of the wall surface of the gas chamber is made of a soft elastic material so that the gas chamber has an inflated state when inhaling air and a concave state when exhaling air; the gas chamber is connected to a branch of the bronchial tree, and the position of the gas chamber and its volume in the inflated state match the corresponding branch end position and its shunt ratio or target shunt ratio; the liquid chamber is connected to the CO2 exhalation device.

10. A cell culture exposure device, characterized in that, It includes a water bath container and a cell culture exposure unit as described in any one of claims 1 to 8 disposed in the water bath container.

11. The cell culture exposure device as described in claim 10, characterized in that, The number of cell culture exposure units is 2 to 10. The distribution location of the cell culture exposure units and the size of their gas chambers are adapted to the location, distribution coefficient and flow rate of the terminal branches of the bronchial tree or the target distribution coefficient and flow rate.

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