Bionic cell culture device, tobacco product detection device and method of using tobacco product detection device

Abstract

This application relates to the interdisciplinary fields of in vitro toxicology evaluation and bioengineering, and in particular to a biomimetic cell culture device, a tobacco product testing device, and a method of using the tobacco product testing device. A biomimetic cell culture device includes: an upper cover assembly, a lower cover assembly, and the upper cover assembly enclosing a chamber; a porous cell culture membrane disposed between the two, dividing the chamber into an upper chamber and a lower chamber; the porous cell culture membrane having a first culture section facing the upper chamber and a second culture section facing the lower chamber; a deformation tube disposed in the chamber and forming an independent gas channel, one side of which abuts against the porous cell culture membrane, and the other side abuts against either the upper or lower cover assembly; the deformation tube is configured such that within its working deformation range, the deformation tube remains in non-contact with the first and second culture sections. The data obtained by the biomimetic cell culture device of this application during tobacco product testing exhibit high physiological relevance and accuracy.

Description

Technical Field

[0001] This application relates to the interdisciplinary fields of in vitro toxicology evaluation and bioengineering, and in particular to a biomimetic cell culture device, a tobacco product testing device, and a method for using the tobacco product testing device. Background Technology

[0002] The biosafety assessment of tobacco products is a core scientific task in product regulation and industrial development. Its fundamental purpose is to systematically evaluate the potential health risks posed by tobacco and its products to users during consumption.

[0003] Existing methods for evaluating the biosafety of tobacco products, especially those involving complex aerosol exposures (such as traditional cigarette smoke and e-cigarette aerosols), have long relied on animal inhalation exposure experiments or simple in vitro cell cultures (such as gas-liquid interface cultures). Animal inhalation exposure experiments suffer from issues such as species differences, ethical controversies, high costs, and low throughput; cells in in vitro cell cultures are in a static culture environment, making it difficult to simulate the complete tissue barrier function of the respiratory tract, the dynamic mucociliary clearance mechanism, and the deposition, dissolution, and absorption processes of aerosols in airway surface fluids, thus limiting the physiological relevance and predictive value of the evaluation results. Summary of the Invention

[0004] Therefore, it is necessary to provide a biomimetic cell culture device, a tobacco product testing device, and a method for using the tobacco product testing device that can solve the above problems.

[0005] To solve the above-mentioned technical problems, this application provides the following technical solution:

[0006] A biomimetic cell culture device, the biomimetic cell culture device comprising:

[0007] Top cover assembly;

[0008] The lower cover plate assembly is sealed to the upper cover plate and forms a cavity with the upper cover plate;

[0009] A porous cell culture membrane is disposed between the upper cover plate assembly and the lower cover plate assembly, dividing the chamber into an upper chamber and a lower chamber. The porous cell culture membrane has a first culture section on the side facing the upper chamber and a second culture section on the side facing the lower chamber. The first culture section and the second culture section are used for culturing cells.

[0010] A deformation tube is disposed in the chamber and forms an independent gas channel for communication with external equipment. One side of the deformation tube abuts against the porous cell culture membrane, and the other side abuts against the upper cover plate assembly or the lower cover plate assembly. The deformation tube can contract / expand radially.

[0011] The deformation tube is configured such that, within its working deformation range, any part of the deformation tube remains in non-contact with the first culture section and the second culture section.

[0012] Understandably, this application, by setting up a porous cell culture membrane including a first culture section and a second culture section, and a deformation tube with independent gas channels, and ensuring that the deformation tube remains non-contact with the first and second culture sections when deformed, allows for cell seeding in the first and second culture sections on both sides of the porous cell culture membrane. The actual inhalation process is simulated by injecting aerosols into the chamber. Simultaneously, the deformation tube deforms due to pressure changes within it, causing the porous cell membrane to deform as well. This mechanical deformation simulates human respiration during cell culture without affecting the cell culture process. Therefore, the combination of bilateral cell culture in the porous cell culture membrane with mechanical deformation to simulate human respiration allows for more accurate simulation of human airways (such as bronchi or alveolar regions) for tobacco product testing, resulting in data with high physiological relevance and accuracy.

[0013] In one embodiment, the upper cover plate assembly is provided with a first receiving groove communicating with the upper chamber, and the lower cover plate assembly is provided with a second receiving groove communicating with the lower chamber at a corresponding position. The first receiving groove and the second receiving groove enclose to form a receiving channel. The outer side of the cell culture membrane is disposed in conjunction with the wall of the first receiving groove or the second receiving groove, and a deformable part is formed at the outer edge of the first culture section. The deformable tube is accommodated in the receiving channel and abuts against the cell culture membrane and the second receiving groove, respectively.

[0014] Understandably, by setting up a receiving channel and a deformation section, the deformation tube is housed in the receiving channel. When the deformation tube deforms, it can push against the deformation section of the porous cell culture membrane, causing the deformation section of the porous cell culture membrane to deform, and causing the first and second culture sections to deform as well. The setting of the deformation section ensures that the deformation tube will not come into contact with the cells in the first and second culture sections when it deforms, and the deformation of the deformation section can cause the first and second culture sections to produce controllable deformation, thereby more accurately simulating the stretching of cells on the porous cell culture membrane caused by human respiration.

[0015] In one embodiment, the deformation tubes are configured as two tubes and are symmetrically arranged along the central axis of the porous cell culture membrane.

[0016] It is understandable that by configuring two deformation tubes and symmetrically arranging them along the central axis of the porous cell culture membrane, forces can be applied evenly from both sides to the first and second culture sections simultaneously, thereby more accurately simulating the stretching of cells on the porous cell culture membrane caused by human respiration.

[0017] In one embodiment, the upper cover assembly includes a first upper cover forming the first receiving groove, the lower cover assembly includes a first lower cover forming the second receiving groove, the first upper cover and the first lower cover are sealed together, and the porous cell culture membrane is disposed between the first upper cover and the first lower cover.

[0018] In one embodiment, the upper cover assembly includes a second upper cover disposed above the first upper cover, and the lower cover assembly includes a second lower cover disposed below the first lower cover.

[0019] The second upper cover plate has a first connection port and a second connection port, the first upper cover plate has a first flow channel communicating with the first connection port and the upper chamber respectively, and the first lower cover plate has a second flow channel communicating with the second connection port and the lower chamber respectively.

[0020] In one embodiment, the first upper cover plate is provided with a first through opening corresponding to the first culture section, and the second upper cover plate is provided with a first observation window corresponding to the first culture section;

[0021] And / or, the first lower cover plate is provided with a second through opening corresponding to the second culture section, and the second lower cover plate is provided with a second observation window corresponding to the second culture section.

[0022] In one embodiment, the second upper cover plate is detachably connected to the first upper cover plate;

[0023] And / or, the second lower cover plate is detachably connected to the first lower cover plate.

[0024] This application also provides the following technical solutions:

[0025] A tobacco product testing device includes a first connector, a second connector, and a biomimetic cell culture device as described in any of the above embodiments.

[0026] In one embodiment, the tobacco product testing device further includes a deformation tube connector and a pressure controller capable of simulating human breathing. The deformation tube connector can be connected to the deformation tube, and the pressure controller is connected to the deformation tube connector and can control the pressure of the deformation tube through the deformation tube connector to cause the deformation tube to deform.

[0027] This application also provides the following technical solutions:

[0028] A method of using the tobacco product testing device provided in the above embodiments includes the following steps:

[0029] (1) Pre-treat the biomimetic cell culture device;

[0030] (2) Inoculate the lower endothelial cells and the upper epithelial cells in the first culture section and the second culture section respectively;

[0031] (3) Conduct aerosol exposure experiments and turn on the air pressure controller to drive the deformation tube to deform in order to simulate the lung environment;

[0032] (4) The porous cell culture membrane was recovered and analyzed.

[0033] Compared to existing technologies, the biomimetic cell culture device, by incorporating a porous cell culture membrane comprising a first and second culture section and a deformation tube with independent gas channels, and by ensuring that the deformation tube remains non-contact with the first and second culture sections during deformation, allows for cell inoculation into both sides of the porous cell culture membrane. It simulates the actual inhalation process by injecting aerosols into the chamber, while simultaneously utilizing pressure changes within the deformation tube to cause deformation, which in turn deforms the porous cell membrane. This mechanical deformation simulates human respiration during cell culture without affecting the cell culture process. Therefore, the combination of bilateral cell culture with mechanical deformation to simulate human respiration allows for more precise simulation of human airways (such as bronchi or alveolar regions) for tobacco product testing, resulting in data with high physiological relevance and accuracy. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application or the conventional technology, the drawings used in the description of the embodiments or the conventional technology will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a schematic diagram of the structure of the tobacco product testing device provided in this application.

[0036] Figure 2 A top view of the tobacco product testing device provided in this application.

[0037] Figure 3 For this application Figure 2 Enlarged view of section AA in the middle.

[0038] Figure 4 An exploded view of the tobacco product testing device provided in this application.

[0039] Figure 5 Another exploded view of the tobacco product testing device provided in this application.

[0040] The component labels are as follows:

[0041] 100. Bionic cell culture device; 10. Top cover assembly; 11. First receiving groove; 111. Receiving channel; 12. First top cover; 121. First flow channel; 122. First through opening; 13. Second top cover; 131. First connection port; 1311. First connection inlet; 1312. First connection outlet; 132. Second connection port; 1321. Second connection inlet; 1322. Second connection outlet; 133. First observation window; 20. Lower cover plate assembly; 21. Chamber; 211. Upper chamber; 212. Lower chamber; 22. Second receiving groove; 23. First lower cover plate; 231. Second flow channel; 232. Second through opening; 24. Second lower cover plate; 241. Second observation window; 242. Positioning protrusion; 243. Positioning groove; 30. Porous cell culture membrane; 31. First culture section; 32. Second culture section; 33. Deformation section; 40. Deformation tube; 41. Gas channel;

[0042] 200. Tobacco product testing device; 201. First connector; 202. Second connector; 203. Deformation tube connector. Detailed Implementation

[0043] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0044] It should be noted that when a component is referred to as being "fixed to" or "set on" another component, it can be directly on the other component or there may be an intermediate component. When a component is considered to be "connected to" another component, it can be directly connected to the other component or there may be an intermediate component present. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and similar expressions used in this application's specification are for illustrative purposes only and do not represent the only possible implementation.

[0045] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0046] In this application, unless otherwise expressly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature and the second feature are in indirect contact through an intermediate medium. Furthermore, "above," "over," and "on top" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0047] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used in this application includes any and all combinations of one or more of the associated listed items.

[0048] Please see Figures 1 to 5This application provides a biomimetic cell culture device 100 for culturing cells and can be applied to fields such as toxicity assessment (e.g., assessing damage to airway cells caused by environmental pollutants, cigarette smoke, etc.), disease mechanism research (for studying the pathophysiological processes of respiratory diseases such as lung injury, lung infection, pulmonary fibrosis, lung tumors, and airway diseases), and drug development and screening (for testing drug efficacy and toxicity indicators, accelerating candidate drug screening). In this application, the biomimetic cell culture device 100 is primarily used for comparative toxicity studies of traditional tobacco smoke and novel electronic cigarette aerosols, product risk assessment, and harm reduction potential verification, providing advanced in vitro technology tools for regulatory science, product development, and safety claims in the tobacco industry.

[0049] Specifically, the biomimetic cell culture device 100 includes an upper cover plate assembly 10, a lower cover plate assembly 20, a porous cell culture membrane 30, and a deformation tube 40. The lower cover plate assembly 20 is sealed to the upper cover plate and forms a chamber 21 with the upper cover plate. The porous cell culture membrane 30 is disposed between the upper cover plate assembly 10 and the lower cover plate assembly 20, dividing the chamber 21 into an upper chamber 211 and a lower chamber 212. The side of the porous cell culture membrane 30 facing the upper chamber 211 has a first culture section 31, and the side of the porous cell culture membrane 30 facing the lower chamber 212 has a... There is a second culture section 32, and the first culture section 31 and the second culture section 32 are used for culturing cells; the deformation tube 40 is disposed in the chamber 21 and forms an independent gas channel 41 for communication with external equipment. One side of the deformation tube 40 abuts against the porous cell culture membrane 30, and the other side abuts against the upper cover plate assembly 10 or the lower cover plate assembly 20. The deformation tube 40 can contract / expand radially; wherein, the deformation tube 40 is configured such that: within its working deformation range, any part of the deformation tube 40 remains in non-contact with the first culture section 31 and the second culture section 32.

[0050] Current cell culture devices typically use gas-liquid interface culture systems such as Transwell chambers. While this model can culture cells on membranes to simulate the epithelial barrier, its exposure to tobacco aerosols usually involves dissolving condensates from tobacco product combustion or atomization (such as tobacco leaf infusion) in the culture medium and adding it in liquid form. This "liquid exposure" mode is fundamentally different from the physiological process of human lung cells directly contacting "gaseous aerosols." The deposition, dissolution, and absorption kinetics of aerosols in airway surface fluids, as well as their interactions with pulmonary surfactant and the mucus layer, cannot be simulated. Furthermore, high concentrations of liquid condensates can easily lead to drastic changes in the physicochemical properties of the culture medium (such as changes in pH and increased osmotic pressure), causing acute cell damage or even death. It is difficult to distinguish between specific toxicity and non-specific damage, resulting in severely insufficient physiological relevance and accuracy of the obtained data. Moreover, the cells in this model are in a static culture state, simply "lying" on the substrate, and cannot simulate the mechanical expansion and contraction of lung cells caused by the breathing movements accompanying smoking. This lack of mechanical signaling leads to discrepancies between cellular stress responses to tobacco aerosols, barrier repair capabilities, and damage pathways and actual in vivo conditions, thus affecting the accurate assessment of chronic effects (such as epithelial-mesenchymal transition and tissue remodeling) under long-term aerosol exposure. This application addresses this issue by setting up a porous cell culture membrane 30 comprising a first culture section 31 and a second culture section 32, and a deformation tube 40 with an independent gas channel 41. When the deformation tube 40 deforms, it remains non-contact with the first and second culture sections 31 and 32. This allows for cell seeding in the first and second culture sections 31 on both sides of the porous cell culture membrane 30, and simulates the actual inhalation process by perfusing aerosols into the chamber 21. Simultaneously, the pressure change within the deformation tube 40 causes it to deform, leading to deformation of the porous cell membrane. This mechanical deformation simulates human respiration during cell culture without affecting the cell culture process. Therefore, the bilateral cell culture of the porous cell culture membrane 30, combined with the mechanical deformation to simulate the structure of human respiration, can more accurately simulate the human airway (such as the bronchus or alveolar region) for tobacco product testing, and the obtained data has high physiological relevance and accuracy.

[0051] like Figures 3 to 5As shown, the upper cover plate assembly 10 is provided with a first receiving groove 11 communicating with the upper chamber 211, and the lower cover plate assembly 20 is provided with a second receiving groove 22 communicating with the lower chamber 212 at a corresponding position. The first receiving groove 11 and the second receiving groove 22 enclose each other to form a receiving channel 111. The outer side of the cell culture membrane is attached to the wall of the first receiving groove 11 or the second receiving groove 22, and a deformation part 33 is formed on the outer edge of the first culture part 31. The deformation tube 40 is accommodated in the receiving channel 111 and abuts against the cell culture membrane and the second receiving groove 22 respectively. Thus, by setting up the receiving channel 111 and the deformation part 33, the deformation tube 40 is housed in the receiving channel 111. When the deformation tube 40 deforms, it can push the deformation part 33 of the porous cell culture membrane 30, causing the deformation part 33 of the porous cell culture membrane 30 to deform, and causing the first culture part 31 and the second culture part 32 to deform. The setting of the deformation part 33 ensures that the deformation tube 40 will not come into contact with the cells of the first culture part 31 and the second culture part 32 when it deforms, and the deformation of the deformation part 33 can cause the first culture part 31 and the second culture part 32 to produce controllable deformation, thereby more accurately simulating the stretching of cells on the porous cell culture membrane 30 by human respiration.

[0052] In one embodiment, the upper cover plate assembly 10 includes a first upper cover plate 12 forming a first receiving groove 11, and the lower cover plate assembly 20 includes a first lower cover plate 23 forming a second receiving groove 22. The first upper cover plate 12 and the first lower cover plate 23 are sealed together, and a porous cell culture membrane 30 is disposed between the first upper cover plate 12 and the first lower cover plate 23.

[0053] Here, the first upper cover plate 12 and the first lower cover plate 23 can be sealed together by means of bonding, bonding or other methods. When replacement is required, the entire first upper cover plate 12, the first lower cover plate 23 and the porous cell culture membrane 30 can be replaced directly.

[0054] In other embodiments, the first upper cover plate 12 and the first lower cover plate 23 can also be connected in a detachable sealed manner by means of snap-fit ​​connection, bolt connection or other means, combined with a sealing ring. In this way, the user can remove the first upper cover plate 12 and the first lower cover plate 23 and replace the porous cell culture membrane 30.

[0055] like Figures 3 to 5As shown, the upper cover assembly 10 also includes a second upper cover 13, which is disposed at the upper end of the first upper cover 12. The lower cover assembly 20 includes a second lower cover 24, which is disposed at the lower end of the first lower cover 23. The second upper cover 13 has a first connection port 131 and a second connection port 132. The first upper cover 12 has a first flow channel 121 that communicates with the first connection port 131 and the upper chamber 211 respectively. The first lower cover 23 has a second flow channel 231 that communicates with the second connection port 132 and the lower chamber 212 respectively.

[0056] In this embodiment, the first connection port 131 includes a first connection inlet 1311 and a first connection outlet 1312, and the second connection port 132 includes a second connection inlet 1321 and a second connection outlet 1322. Cell nutrient solution and flue gas aerosol can enter the upper chamber 211 through the first connection inlet 1311 and flow out through the first connection outlet 1312. Cell nutrient solution and flue gas aerosol can enter the lower chamber 212 through the second connection inlet 1321 and flow out through the second connection outlet 1322.

[0057] Furthermore, the first upper cover plate 12 is provided with a first through opening 122 corresponding to the first culture section 31, and the second upper cover plate 13 is provided with a first observation window 133 corresponding to the first culture section 31. By providing the first through opening 122 and the first observation window 133, it is convenient for users to observe the first culture section 31 during and after the detection process, thereby improving the intuitiveness of the cell culture device detection.

[0058] In one embodiment, the first lower cover plate 23 is provided with a second through opening 232 corresponding to the second culture section 32, and the second lower cover plate 24 is provided with a second observation window 241 corresponding to the second culture section 32. By providing the second through opening 232 and the second observation window 241, it is convenient for users to observe the second culture section 32 during and after the detection process, thereby improving the intuitiveness of the cell culture device detection.

[0059] In one embodiment, the second upper cover plate 13 is detachably connected to the first upper cover plate 12. Here, the connection method between the second upper cover plate 13 and the first upper cover plate 12 can be screw connection, snap-fit ​​connection, bolt connection, clamp fixation or magnetic connection, etc.

[0060] In one embodiment, the second lower cover plate 24 is detachably connected to the first lower cover plate 23. Here, the connection method between the second upper cover plate 13 and the first upper cover plate 12 can be referred to the connection method between the second upper cover plate 13 and the first upper cover plate 12, and will not be described in detail here.

[0061] In one embodiment, the second upper cover plate 13 and the first upper cover plate 12, and the second lower cover plate 24 and the first lower cover plate 23 are positioned by providing a positioning protrusion 242 on one side and a positioning groove 243 on the other side. By providing the mating structure of the positioning protrusion 242 and the positioning groove 243, the second upper cover plate 13 and the second lower cover plate 24 can be precisely guided and mechanically limited during installation, enabling quick and accurate installation of the second upper cover plate 13 and the second lower cover plate 24.

[0062] Preferably, the positioning protrusion 242 is configured as a hemispherical protrusion and the positioning groove 243 is configured as a hemispherical groove. By setting the hemispherical protrusion and the hemispherical groove for positioning, the contact surface is optimized into a hemispherical protrusion and a hemispherical groove, which can reduce friction, optimize stress distribution, make the installation of the second upper cover plate 13 and the second lower cover plate 24 smoother, and reduce the wear of the positioning protrusion 242 to extend its service life.

[0063] like Figures 3 to 5 As shown, two deformation tubes 40 are configured and symmetrically arranged along the central axis of the porous cell culture membrane 30. By configuring two deformation tubes 40 symmetrically along the central axis of the porous cell culture membrane 30, forces can be applied simultaneously and evenly from both sides to the first culture section 31 and the second culture section 32, thereby more accurately simulating the stretching of cells on the porous cell culture membrane 30 caused by human respiration. In other embodiments, to achieve simulation of human respiration in different areas or regions, multiple deformation tubes 40 can be configured, such as three or four, or in other shapes such as rings or arcs.

[0064] This application also provides the following technical solutions:

[0065] A tobacco product testing device 200 includes a first connector 201, a second connector 202, and a biomimetic cell culture device 100 as described in any of the above embodiments.

[0066] like Figure 4 As shown in this embodiment, two first connectors 201 and two second connectors 202 are respectively provided. The two first connectors 201 are respectively connected to the first connection inlet 1311 and the first connection outlet 1312, and the two second connectors 202 are respectively connected to the second connection inlet 1321 and the second connection outlet 1322.

[0067] In one embodiment, the first connector 201 and the second connector 202 are configured as standardized Luer quick-connect interfaces, which are directly compatible with mainstream flue gas generating equipment, can improve the stability of aerosol transmission efficiency, and improve the uniformity of flue gas aerosol distribution in the chamber 21 of the biomimetic cell culture device 100.

[0068] In one embodiment, the tobacco product detection device 200 further includes a deformation tube connector 203 and a pressure controller (not shown) capable of simulating human breathing. The deformation tube connector 203 can be connected to the deformation tube 40, and the pressure controller is connected to the deformation tube connector 203 and can control the pressure of the deformation tube 40 through the deformation tube connector 203 to drive the deformation tube 40 to deform.

[0069] This application also provides the following technical solutions:

[0070] A method of using a tobacco product testing device 200 includes the following steps:

[0071] (1) Pre-treatment of the biomimetic cell culture device 100;

[0072] (2) Lower endothelial cells and upper epithelial cells were seeded in the first culture section 31 and the second culture section 32, respectively;

[0073] (3) Conduct aerosol exposure experiment and turn on the air pressure controller to drive the deformation tube 40 to deform to simulate the lung environment;

[0074] (4) The porous cell culture membrane 30 was recovered and analyzed.

[0075] The specific operating procedure of the tobacco product testing device 200 is illustrated below:

[0076] Phase 1: Pretreatment and seeding of lower endothelial cells using the biomimetic cell culture device 100.

[0077] The goal of this stage is to construct a fused layer of pulmonary microvascular endothelial cells in the second culture section 32 of the porous cell culture membrane 30, serving as the base for a biological barrier.

[0078] 1. Preparation of the biomimetic cell culture device 100 and opening of the lower chamber 212:

[0079] (1) Remove the second lower cover plate 24. At this time, the second culture section 32 of the porous cell culture membrane 30 is exposed through the second through opening 232 of the first lower cover plate 23.

[0080] (2) Check whether the surface of the porous cell culture membrane 30 is flat and clean.

[0081] 2. Membrane surface modification:

[0082] (1) Place the biomimetic cell culture device 100 in the clean bench and use a micropipette to directly add 50-100 μL of extracellular matrix solution (such as 0.1 mg / mL collagen IV solution) to the second culture section 32, ensuring that the solution covers the entire second culture section 32 (such as a Φ14 mm circular area).

[0083] (2) Transfer the biomimetic cell culture device 100 to a 4°C refrigerator and let it stand overnight (about 16 hours) to allow the matrix proteins to fully adsorb. The next day, aspirate the excess solution, gently rinse twice with sterile PBS, and set aside.

[0084] 3. Endothelial cell seeding and static culture:

[0085] (1) The pulmonary microvascular endothelial cells were resuspended in complete endothelial cell culture medium and the cell density was adjusted to 1.0 × 10⁻⁶. 6 cells / mL.

[0086] (2) Place the biomimetic cell culture device 100 at an angle, use a pipette to draw 50 μL of cell suspension, and slowly drop it onto the center of the second culture section 32 of the coated porous cell culture membrane 30, relying on the surface tension of the liquid to allow it to spread naturally and cover the entire modified area.

[0087] (3) Place the biomimetic cell culture device 100 flat in a sterile culture dish and transfer it to a cell culture incubator (37°C, 5% CO2, 95% humidity) for static culture. The cells will complete the initial attachment within 2-4 hours.

[0088] (4) After standing for 4 hours, slowly inject pre-warmed complete endothelial cell culture medium into the second connection port 132 of the biomimetic cell culture device 100 until the entire first lower cover plate 23 is filled. Then connect the micro-perfusion pump and start continuous perfusion at a very low flow rate (e.g., 0.5 μL / min) to maintain endothelial cell viability. Continue culturing for 24-48 hours until it is confirmed through the second observation window 241 (at this time, observation can be performed by temporarily placing the cover plate or using an inverted microscope) that the endothelial cells have formed a dense and continuous monolayer.

[0089] Second stage: upper epithelial cell seeding and biomimetic cell culture device 100 sealed.

[0090] The goal of this stage is to seed alveolar epithelial cells in the second culture section 32 and complete the sealed assembly of the biomimetic cell culture device 100.

[0091] 1. The biomimetic cell culture device 100 is flipped and the upper chamber 211 is opened:

[0092] (1) After confirming the formation of the endothelial layer, stop the perfusion of the lower chamber 212. Under aseptic conditions, rotate the biomimetic cell culture device 100 180 degrees and fix it.

[0093] (2) Open the second upper cover plate 13. At this time, the first culture section 31 is exposed through the first through opening 122.

[0094] 2. Epithelial cell inoculation:

[0095] (1) Resuspend human alveolar epithelial cells (such as A549 cells or primary human alveolar epithelial cells) in a special gas-liquid interface culture medium with a density of 2.0 × 10⁻⁶. 6 cells / mL.

[0096] (2) Add 100 μL of cell suspension directly to the first culture section 31, ensuring that the same planting area as the second culture section 32 is covered.

[0097] (3) Place the biomimetic cell culture device 100 back into the incubator and let it stand for 2-3 hours to allow the epithelial cells to initially attach.

[0098] 3. Final sealing and gas-liquid interface establishment of the biomimetic cell culture device 100:

[0099] (1) After the cells attach, remove excess liquid from the surface of the first culture section 31. Install the second upper cover plate 13 onto the first upper cover plate 12.

[0100] (2) Flip the bionic cell culture device 100 and install the second lower cover plate 24.

[0101] (3) Sterile humidified air (5% CO2) was introduced into the first upper cover plate 12 through a small balancing vent (not shown, approximately 0.5 mm in diameter) on the side of the first connector 201, while the perfusion flow rate of the culture medium in the lower chamber 212 was adjusted to 2 μL / min. This operation allowed the liquid on the membrane surface to be slowly evaporated / absorbed, and after approximately 24 hours, an air-liquid interface was successfully established at the tip of the alveolar epithelial cells. The epithelial cells continued to differentiate and mature under these conditions for 5-7 days.

[0102] Phase 3: Combined experiment of aerosol exposure and dynamic mechanical stimulation.

[0103] Once the transmembrane resistance value stabilizes (indicating that the barrier function is mature), exposure experiments can begin.

[0104] 1. System Connection:

[0105] (1) Aerosol exposure connection: The first connector 201 of the bionic cell culture device 100 is directly connected to the exposure chamber outlet of a program-controlled electronic cigarette aerosol generator (such as the German Vitrocell VC 10 smoking machine) through a Teflon tube, and the gas connection is sealed.

[0106] (2) Mechanical stimulation connection: Connect the deformation tube 40 to the deformation tube connector 203 and connect it to an external programmable air pressure controller through a hose.

[0107] (3) Ensure that the perfusion system of the lower chamber 212 continues to work.

[0108] 2. Experimental Operation:

[0109] (1) An external air pressure controller is set up to output a sinusoidal air pressure signal to drive the deformation tube 40 of the bionic cell culture device 100 to undergo periodic deformation, and apply a mechanical stimulus of 10% tensile strain and 0.2 Hz (simulating calm breathing) to the double-layer cells through the deformation part 33.

[0110] (2) Start the electronic cigarette device and set the smoking machine to the "Canadian deep inhalation" standard mode. The generated aerosol is introduced into the first upper cover plate 12 through the first connector 201 to directly expose the alveolar epithelial cells. The exposure cycle can be set as follows: 2 seconds of inhalation, 58 seconds of interval, and repeated.

[0111] (3) Throughout the exposure period (e.g., 6 hours), cell morphology is photographed at regular intervals using a live-cell imaging system through the first observation window 133 and the second observation window 241. At the same time, the effluent from the lower chamber 212 is collected for subsequent analysis of inflammatory factors or toxic markers that have crossed the barrier.

[0112] Phase 4: Endpoint sample recovery and analysis.

[0113] After the exposure experiment, flexible endpoint analysis can be performed without damaging the main body of the biomimetic cell culture device 100.

[0114] 1. In-situ detection:

[0115] (1) Barrier integrity damage was assessed by measuring the change in transmembrane resistance after exposure through a microelectrode interface (not shown) pre-integrated on the cover plate.

[0116] (2) Inject live cell dye (such as Calcein-AM / PI) into chamber 21 through injection hole (not shown in the figure), and perform in situ fluorescence imaging directly through observation window (2, 10) to assess cell viability.

[0117] 2. Sample recovery after opening the lid:

[0118] (1) Disconnect all external connections. Open the second upper cover 13 and the second lower cover 24.

[0119] (2) Epithelial cell recovery: 100 μL of trypsin digestion solution was added to the first culture section 31 through the first through opening 122, incubated at 37°C for 5 minutes, and the cell suspension was aspirated after gentle pipetting, centrifuged and collected for subsequent molecular biological analysis such as qPCR and Western Blot.

[0120] (3) Endothelial cell recovery: The cells in the second culture section 32 were digested and recovered using the same method.

[0121] (4) In situ analysis of membrane surface: If immunofluorescence staining is required, fixative, perforation solution and antibody can be added directly to the cells on the porous cell culture membrane 30 after opening the lid for staining. After staining, the entire porous cell culture membrane 30 can be carefully cut off from the main body of the bionic cell culture device 100, sealed and observed under a microscope.

[0122] The above testing methods can be selected by the laboratory personnel according to their needs.

[0123] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0124] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Therefore, the patent protection scope of this application should be determined by the appended claims.

Claims

1. A biomimetic cell culture device, characterized in that, The biomimetic cell culture device (100) includes: Top cover assembly (10); The lower cover plate assembly (20) is sealed to the upper cover plate and forms a cavity (21) with the upper cover plate. A porous cell culture membrane (30) is disposed between the upper cover plate assembly (10) and the lower cover plate assembly (20), and divides the chamber (21) into an upper chamber (211) and a lower chamber (212). The porous cell culture membrane (30) has a first culture section (31) on the side facing the upper chamber (211) and a second culture section (32) on the side facing the lower chamber (212). The first culture section (31) and the second culture section (32) are used for culturing cells. A deformation tube (40) is disposed in the chamber (21) and forms an independent gas channel (41) for communication with external equipment. One side of the deformation tube (40) abuts against the porous cell culture membrane (30), and the other side abuts against the upper cover plate assembly (10) or the lower cover plate assembly (20). The deformation tube (40) can contract / expand radially. The deformation tube (40) is configured such that any part of the deformation tube (40) remains in non-contact with the first culture section (31) and the second culture section (32) within its working deformation range.

2. The biomimetic cell culture device according to claim 1, characterized in that, The upper cover assembly (10) is provided with a first receiving groove (11) communicating with the upper chamber (211), and the lower cover assembly (20) is provided with a second receiving groove (22) communicating with the lower chamber (212) at a corresponding position. The first receiving groove (11) and the second receiving groove (22) enclose to form a receiving channel (111). The outer side of the cell culture membrane is attached to the wall of the first receiving groove (11) or the second receiving groove (22), and a deformation part (33) is formed on the outer edge of the first culture part (31). The deformation tube (40) is accommodated in the receiving channel (111) and abuts against the cell culture membrane and the second receiving groove (22) respectively.

3. The biomimetic cell culture device according to claim 2, characterized in that, The deformation tubes (40) are configured as two tubes and are symmetrically arranged along the central axis of the porous cell culture membrane (30).

4. The biomimetic cell culture device according to claim 2, characterized in that, The upper cover assembly (10) includes a first upper cover (12) that forms the first receiving groove (11), and the lower cover assembly (20) includes a first lower cover (23) that forms the second receiving groove (22). The first upper cover (12) and the first lower cover (23) are sealed together, and the porous cell culture membrane (30) is disposed between the first upper cover (12) and the first lower cover (23).

5. The biomimetic cell culture device according to claim 4, characterized in that, The upper cover assembly (10) includes a second upper cover (13), which is disposed at the upper end of the first upper cover (12), and the lower cover assembly (20) includes a second lower cover (24), which is disposed at the lower end of the first lower cover (23). The second upper cover plate (13) has a first connection port (131) and a second connection port (132), the first upper cover plate (12) has a first flow channel (121) that communicates with the first connection port (131) and the upper chamber (211) respectively, and the first lower cover plate (23) has a second flow channel (231) that communicates with the second connection port (132) and the lower chamber (212) respectively.

6. The biomimetic cell culture device according to claim 5, characterized in that, The first upper cover plate (12) is provided with a first through opening (122) corresponding to the first culture section (31), and the second upper cover plate (13) is provided with a first observation window (133) corresponding to the first culture section (31). And / or, the first lower cover plate (23) is provided with a second through opening (232) corresponding to the second culture section (32), and the second lower cover plate (24) is provided with a second observation window (241) corresponding to the second culture section (32).

7. The biomimetic cell culture device according to claim 5, characterized in that, The second upper cover plate (13) is detachably connected to the first upper cover plate (12); And / or, the second lower cover plate (24) is detachably connected to the first lower cover plate (23).

8. A tobacco product testing device, characterized in that, It includes a first connector (201), a second connector (202), and a biomimetic cell culture device (100) as described in any one of claims 1-7.

9. The tobacco product testing device according to claim 8, characterized in that, The tobacco product testing device (200) also includes a deformation tube connector (203) and a pressure controller that can simulate human breathing. The deformation tube connector (203) can be connected to the deformation tube (40), and the pressure controller is connected to the deformation tube connector (203) and can control the pressure of the deformation tube (40) through the deformation tube connector (203) to drive the deformation tube (40) to deform.

10. A method of using the tobacco product testing device according to any one of claims 8-9, characterized in that, The usage method includes the following steps: (1) Pre-treat the biomimetic cell culture device (100); (2) Lower endothelial cells and upper epithelial cells are seeded in the first culture section (31) and the second culture section (32), respectively; (3) Conduct aerosol exposure experiment and turn on the air pressure controller to drive the deformation tube (40) to deform to simulate the lung environment; (4) The porous cell culture membrane (30) was recovered and analyzed.

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