Device for biologically simulating flue gas exposure as well as use method and application thereof

Abstract

The invention discloses a device for biologically simulating flue gas exposure as well as a use method and application thereof. The device for biologically simulating smoke exposure comprises a cell culture layer, a porous membrane, a fluid channel layer, a cell exposure box and a water bath box. The device for biologically simulating smoke exposure can simulate a three-dimensional microenvironment and dynamic blood flow of a gas-liquid interface of a respiratory tract in a human body, can realize full contact of human lung epithelial cells with air and smoke, can simulate the intensity and frequency of smoke stimulation on lungs in the human body during smoking, improves the accuracy of smoke exposure evaluation, and has a good application prospect. The method can be applied to multiple fields of environmental pollution detection, drug evaluation, clinical test safety evaluation and the like.

Description

Technical Field

[0001] The present invention belongs to the fields of medicine and medical devices, and relates to a device for biomimetic smoke exposure, its use method and application. Background Art

[0002] A microfluidic chip, also known as a Lab on a Chip, usually integrates a series of operations such as sample preparation, reaction, separation, and detection in the analysis process of biomedicine on a single chip to complete an integrated system function. Organoid technology is a culture technology that mimics the microenvironment of tissues and organs in the human body in vitro. Combining organoid technology with microfluidic technology can integrate in vitro biological simulation onto a tiny chip platform for pharmacological and toxicological research, which has the characteristics of low cost and high throughput. Smoke analyzers are widely used in the fields of industry and environmental monitoring and can accurately analyze various stimulant components in smoke, but from a biological perspective, there is a lack of a more suitable evaluation device to reflect the human body's response to smoke exposure.

[0003] Therefore, there is an urgent need to provide a device and method for mimicking smoke exposure from a biological perspective. Summary of the Invention

[0004] In view of the deficiencies of the prior art and the actual needs, the present invention provides a device for biomimetic smoke exposure, its use method and application, aiming to solve the inaccuracy of the results brought by using cells cultured in well plates or non-human animals in the existing smoke exposure assessment process. Using a device for biomimetic smoke exposure that can simulate the cigarette smoking process can improve the accuracy of smoke exposure assessment. When performing detection, only a low dose of reagent and a small amount of cells are required to carry out cell culture and exposure in a microscale and high-throughput chip, reducing costs and improving efficiency.

[0005] To achieve the object of the present invention, the following technical solutions are adopted:

[0006] In a first aspect, the present invention provides a device for biomimetic smoke exposure, which includes a microfluidic chip and a smoke evaluation device; the microfluidic chip includes a cell culture layer 1, a porous membrane 2, and a fluid channel layer 3; the smoke evaluation device includes a cell exposure box 4 and a water bath box 5;

[0007] The cell culture layer 1 includes a fluid inlet 101, cell culture wells 102, and a fluid outlet 103;

[0008] The fluid channel layer 3 includes a fluid channel 301;

[0009] The cell exposure box 4 includes an exposure box air inlet 401, an exposure box air outlet 402, an exposure box lid 403, and a chip mounting platform 404;

[0010] The water bath box 5 includes a water bath box air inlet 501, a water bath box air outlet 502, a water bath box cover 503, a water bath box water inlet 504 and a water bath box water outlet 505.

[0011] The device for biomimetic smoke exposure of the present invention can simulate the three-dimensional microenvironment and dynamic blood flow of the air-liquid interface in the human respiratory tract, can achieve full contact between human lung epithelial cells and air and smoke, simulate the intensity and frequency of lung irritation by smoke in the human body during smoking, improve the accuracy of smoke exposure assessment, and can be applied to multiple fields such as environmental pollution detection, drug evaluation, and clinical trial safety assessment.

[0012] In the present invention, the microfluidic chip is connected to a micro-injection pump device through a PTFE capillary, and a fluid with a specific flow rate can be accurately supplied to the fluid channel 301 through the micro-injection pump.

[0013] In the present invention, a syringe for injecting reagents and a container for recovering excess reagents or waste liquid are respectively connected before and after the fluid channel 301 of the microfluidic chip.

[0014] In the present invention, the cell culture layer 1 and the fluid channel layer 3 can be obtained by casting polydimethylsiloxane with a copper mold and heating and curing.

[0015] Preferably, the microfluidic chip is encapsulated by a glass clamp.

[0016] Preferably, the cell culture holes 102 on the cell culture layer 1 are arranged horizontally and vertically in parallel; the diameter of the cell culture holes 102 is 1.5 - 2.5 mm (such as 1.5 mm, 2 mm or 2.5 mm), the depth is 1.5 - 2.5 mm (such as 1.5 mm, 2 mm or 2.5 mm), and the hole spacing is 4 - 6 mm (such as 4 mm, 5 mm or 6 mm).

[0017] Preferably, the pore diameter of the porous membrane 2 is 0.8 - 8 μm (such as 0.8 μm, 1 μm, 2 μm, 6 μm, 7 μm or 8 μm).

[0018] Preferably, the material of the porous membrane 2 includes polycarbonate.

[0019] In the present invention, the porous membrane 2 completely covers the cell culture layer 1 and the fluid channel layer 3 and fits tightly with them, without gaps and leaks.

[0020] Preferably, the fluid channel 301 is an S-shaped channel, and the width of the S-shaped channel is 0.2 - 0.4 mm, and the depth is 0.05 - 0.2 mm (such as 0.05 mm, 0.1 mm or 0.2 mm).

[0021] Preferably, the materials of the cell culture layer 1 and the fluid channel layer 3 include polydimethylsiloxane and / or polymethyl methacrylate.

[0022] Preferably, the external length of the cell exposure box 4 is 120 - 140 mm (such as 120 mm, 130 mm or 140 mm), the external width is 90 - 110 mm (such as 90 mm, 100 mm or 110 mm), and the external height is 60 - 62 mm (such as 60 mm, 61 mm or 62 mm). The internal length of the cell exposure box 4 is 105 - 115 mm (such as 105 mm, 110 mm or 115 mm), the internal width is 75 - 85 mm (such as 75 mm, 80 mm or 85 mm), and the internal height is 38 - 42 mm (such as 38 mm, 40 mm or 42 mm). A mesh plate is provided inside the cell exposure box 4. The thickness of the mesh plate is 2 - 4 mm (such as 2 mm, 3 mm or 4 mm), and the pore diameter is 8 - 12 mm (such as 8 mm, 10 mm or 12 mm).

[0023] Preferably, the pore diameters of the exposure box air inlet 401 and the exposure box air outlet 402 are 14 - 16 mm (such as 14 mm, 15 mm or 16 mm).

[0024] Preferably, the external length of the water bath box 5 is 225 - 235 mm (such as 225 mm, 230 mm or 235 mm), the external width is 195 - 205 mm (such as 195 mm, 200 mm or 205 mm), and the external height is 205 - 215 mm (such as 205 mm, 210 mm or 215 mm). The internal length of the water bath box 5 is 205 - 215 mm (such as 205 mm, 210 mm or 215 mm), the internal width is 175 - 185 mm (such as 175 mm, 180 mm or 185 mm), and the internal height is 185 - 195 mm (such as 185 mm, 190 mm or 195 mm).

[0025] In a second aspect, the present invention provides a method for using the device for biomimetic flue gas exposure described in the first aspect. The method includes: injecting a fluorescent probe into the cell culture well 102 of the device for biomimetic flue gas exposure described in the first aspect to label cells, scanning the cell culture well 102 using a fluorescence microscope, analyzing the types and contents of the cells labeled with the fluorescent probe through fluorescence intensity, digesting the cells in the cell culture well 102 with trypsin and collecting the cells, and extracting RNA for sequencing analysis.

[0026] The cell culture layer 1, the porous membrane 2 and the fluid channel layer 3 are assembled into a microfluidic chip. The microfluidic chip for culturing lung epithelial cells is placed in a cell exposure box 4, and the cell exposure box 4 is placed in a water bath box 5. The exposure box air inlet 401 and the exposure box air outlet 402 are respectively communicated with the water bath box air inlet 501 and the water bath box air outlet 502. The water bath box water inlet 504 is connected to sterile distilled water. The water inlet 504 is opened and the water outlet 505 is closed to allow sterile distilled water to flow into the lower part of the water bath box, and cigarette smoking fumes are introduced into the water bath box air inlet 501.

[0027] In a third aspect, the present invention provides the use of the device for biomimetic smoke exposure described in the first aspect in smoke exposure assessment, environmental pollution detection or drug assessment.

[0028] Preferably, the smoke exposure assessment includes the cytotoxicity assessment and biomarker assessment of the smoke.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The device for biomimetic smoke exposure of the present invention can simulate the three-dimensional microenvironment and dynamic blood flow of the air-liquid interface in the human respiratory tract, can culture lung epithelial cells in a high-throughput manner and be used to simulate the airway-liquid interface, realize the full contact of human lung epithelial cells with air and smoke, can simulate the intensity and frequency of the smoke stimulation on the human lungs during smoking, is beneficial to the smoke exposure assessment at the biological level, improves the accuracy of the smoke exposure assessment. In addition, it can also be applied to multiple fields such as environmental pollution detection, drug assessment, and clinical trial safety assessment. Description of the Drawings

[0031] Figure 1 It is a schematic top view of the structure of the microfluidic chip of the present invention;

[0032] Figure 2 It is a schematic diagram of the structure of the microfluidic chip of the present invention;

[0033] Figure 3 It is an assembly schematic diagram of the main body of the microfluidic chip of the present invention;

[0034] Figure 4 It is a schematic diagram of the cell exposure box on the inner layer of the smoke assessment device of the present invention;

[0035] Figure 5 It is a schematic diagram of the water bath box on the outer layer of the smoke assessment device of the present invention;

[0036] Figure 6 It is an assembly schematic diagram of the smoke assessment device of the present invention;

[0037] Among them, 1 - cell culture layer, 2 - porous membrane, 3 - fluid channel layer, 101 - fluid inlet, 102 - cell culture well, 103 - fluid outlet, 301 - fluid channel, 4 - cell exposure box, 401 - exposure box air inlet, 402 - exposure box air outlet, 403 - exposure box lid, 404 - chip mounting platform, 5 - water bath box, 501 - water bath box air inlet, 502 - water bath box air outlet, 503 - water bath box lid, 504 - water bath box water inlet, 505 - water bath box water outlet. Detailed implementation manners

[0038] To further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments and the accompanying drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0039] For those technical or conditions not specified in the embodiments, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular channels of commercial purchase.

[0040] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, in which the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions from beginning to end. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as limiting the present invention. In the description of the present invention, it should be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device, element or structure referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as limiting the present invention.

[0041] In addition, the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the present invention, "a plurality of" means two or more, unless otherwise specifically defined.

[0042] In the present invention, unless otherwise clearly specified and limited, terms such as "installation", "connection", "connection", "communication", etc. should be understood in a broad sense. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0043] It should also be understood that the terms used in the specification of the present invention are merely for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the specification of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include the plural forms.

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the scope of protection of the present invention.

[0045] Many specific details are set forth in the following description in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0046] Embodiment 1

[0047] This embodiment provides a device for biomimetic flue gas exposure, and the device for biomimetic flue gas exposure includes a microfluidic chip and a flue gas evaluation device.

[0048] The microfluidic chip is assembled by three layers of chip structures, namely the cell culture layer 1 on the top layer, the porous membrane 2 and the fluid channel layer 3. A reagent fluid inlet 101, a fluid channel 301, a fluid outlet 103, and 4×4 arranged cell culture wells 102 are provided on the microfluidic chip. The diameter of the cell culture well 102 is 2 mm, the depth is 2 mm, and the hole pitch is 5 mm. The fluid channel 301 is an S-shaped channel, and the width of the S-shaped channel is 0.3 mm and the depth is 0.1 mm. The fluid inlet 101 and the fluid outlet 103 are located on the cell culture layer 1, aiming to connect the closed fluid channel 301 with the capillary for adding reagents from the outside. The cell culture well 102 is an open pore channel both above and below in the cell culture layer 1, so that when the microfluidic chip is assembled, the cell culture well 102 can participate in the material exchange with the fluid channel 301 through the porous membrane 2 and the addition of cells, culture medium or drugs from the outside at the same time.

[0049] Preparation of the microfluidic chip: The upper and lower structures of the microfluidic chip, namely the cell culture layer 1 and the fluid channel layer 3, are prepared by pouring polydimethylsiloxane (PDMS) into an inverse mold and baking it to set. The cured PDMS chip is peeled off, holes are drilled in the upper chip, i.e., the cell culture layer 1. After cleaning it with nitrogen, the chip is assembled. The polycarbonate porous membrane is soaked in an aqueous solution of 3-aminopropyltriethoxysilane (APTES). The microfluidic chip is placed in a plasma cleaner for cleaning. The structures of the microfluidic chip are sequentially bonded together, baked, cleaned with nitrogen, placed in a cell culture dish, and 75% ethanol is poured in and vacuumed to remove the air inside the microfluidic chip. The microfluidic chip is inspected. The fabricated microfluidic chip is placed under an inverted microscope, and the details of the microstructures and microchannels are observed to ensure that its microstructures meet the design requirements.

[0050] As described above, after adding cells from the outside to the upper cell culture wells 102, culture medium, drugs or stimulants can be given to the cell culture wells 102 or the fluid channels 301, and the operation method is not uniquely limited. Cells can be cultured and drugs or stimulants can be given for stimulation in multiple cell culture wells 102 simultaneously, giving play to the advantages of high throughput and low cost.

[0051] A micro-injection pump can be externally connected to the fluid inlet 101, and a container for recovering reagents can be externally connected to the fluid outlet 103.

[0052] After successfully culturing lung epithelial cells in the microfluidic chip, the microfluidic chip is placed into the cell exposure chamber 4 of the flue gas evaluation device. The upper and lower parts of the chamber are provided with an exposure chamber air inlet 401 and an exposure chamber air outlet 402. The exposure chamber air inlet 401 is located on the exposure chamber cover 403. A chip mounting platform 404 with pores is arranged in the exposure chamber for mounting the chip. After placing the microfluidic chip, the cell exposure chamber 4 is fixed in the water bath 5. The air inlet and air outlet of the exposure chamber are connected through the water bath air inlet 501 and the water bath air outlet 502. The water bath 5 is provided with a water bath cover 503, and the water bath air inlet 501 is located on the cover. The lower part of the water bath 5 is provided with a water bath air outlet 502, a water bath water inlet 504, and a water bath water outlet 505. Among them, the external length of the cell exposure chamber 4 is 130 mm, the external width is 100 mm, and the external height is 61 mm. The internal length of the cell exposure chamber 4 is 110 mm, the internal width is 80 mm, and the internal height is 40 mm. A mesh plate is arranged inside the cell exposure chamber 4. The thickness of the mesh plate is 3 mm, and the pore diameter is 10 mm. The pore diameters of the exposure chamber air inlet 401 and the exposure chamber air outlet 402 are 15 mm. The external length of the water bath 5 is 230 mm, the external width is 200 mm, and the external height is 210 mm. The internal length of the water bath 5 is 210 mm, the internal width is 180 mm, and the internal height is 190 mm. The porous membrane 2 is pretreated with APTES and then sealed at 80 °C after being cleaned with a cation cleaning instrument together with the cell culture layer 1 and the fluid channel layer 3 to ensure the sealing performance.

[0053] The pore size of the porous membrane 2 is set to 0.8 μm to ensure that the cells are located in the cell culture layer 1 and can carry out normal material exchange with the fluid channel layer 3. The porous membrane 2 uses polycarbonate (PC) material, and the cell culture layer 1 and the fluid channel layer 3 use PDMS material.

[0054] Example 2

[0055] This example provides a device for biomimetic flue gas exposure. The device for biomimetic flue gas exposure includes a microfluidic chip and a flue gas evaluation device.

[0056] The microfluidic chip is assembled from three-layer chip structures, namely the cell culture layer 1 on the top layer, the porous membrane 2, and the fluid channel layer 3. On the microfluidic chip, there are provided a reagent fluid inlet 101, a fluid channel 301, a fluid outlet 103, and 4×4 arranged cell culture wells 102. The diameter of the cell culture well 102 is 1.5 mm, the depth is 1.5 mm, and the well pitch is 4 mm. The fluid channel 301 is an S-shaped channel, and the width of the S-shaped channel is 0.2 mm and the depth is 0.05 mm. The fluid inlet 101 and the fluid outlet 103 are located on the cell culture layer 1, aiming to connect the closed fluid channel 301 with the capillary for adding reagents from the outside. The cell culture well 102 is an open channel both above and below in the cell culture layer 1, so that when the microfluidic chip is assembled, the cell culture well 102 can simultaneously participate in the material exchange with the fluid channel 301 through the porous membrane 2 and add cells, culture medium or drugs from the outside.

[0057] Preparation of the microfluidic chip: The upper and lower structures of the microfluidic chip, namely the cell culture layer 1 and the fluid channel layer 3, are prepared by 3D printing. Using polymethyl methacrylate (PMMA) material, after being cleaned with nitrogen, it is assembled with a polycarbonate (PC) porous membrane by a chip fixture. The assembled chip is placed in a cell culture dish, and 75% ethanol is poured in and vacuumed to remove the air inside the microfluidic chip. The microfluidic chip is inspected. The fabricated microfluidic chip is placed under an inverted microscope, and the details of the microstructures and microchannels are observed to ensure that its microstructures meet the design requirements.

[0058] As described above, after adding cells from the outside to the upper-layer cell culture well 102, culture medium, drugs or stimulants can be given to the cell culture well 102 or the fluid channel 301, and the operation method is not uniquely limited. Cells can be cultured and drugs or stimulants can be given for stimulation in multiple cell culture wells 102 simultaneously, giving play to the advantages of high throughput and low cost.

[0059] A micro-injection pump can be externally connected to the fluid inlet 101, and a container for recovering reagents can be externally connected to the fluid outlet 103.

[0060] After successfully culturing lung epithelial cells in the microfluidic chip, place the microfluidic chip into the cell exposure chamber 4 of the flue gas evaluation device. The upper and lower parts of the chamber are provided with an exposure chamber air inlet 401 and an exposure chamber air outlet 402. The exposure chamber air inlet 401 is located on the exposure chamber cover 403. A chip mounting platform 404 with pores is provided in the exposure chamber for mounting the chip. After placing the microfluidic chip, fix the cell exposure chamber 4 in the water bath 5. Connect the air inlet and air outlet of the exposure chamber through the water bath air inlet 501 and the water bath air outlet 502. The water bath 5 is provided with a water bath cover 503, and the water bath air inlet 501 is located on the cover. The lower part of the water bath 5 is provided with a water bath air outlet 502, a water bath water inlet 504, and a water bath water outlet 505. Among them, the external length of the cell exposure chamber 4 is 120 mm, the external width is 90 mm, and the external height is 60 mm. The internal length of the cell exposure chamber 4 is 105 mm, the internal width is 75 mm, and the internal height is 38 mm. A mesh plate is provided inside the cell exposure chamber 4. The thickness of the mesh plate is 2 mm, and the pore diameter is 8 mm. The pore diameters of the exposure chamber air inlet 401 and the exposure chamber air outlet 402 are 14 mm. The external length of the water bath 5 is 225 mm, the external width is 195 mm, and the external height is 205 mm. The internal length of the water bath 5 is 205 mm, the internal width is 175 mm, and the internal height is 185 mm.

[0061] The upper and lower parts of the microfluidic chip body are encapsulated by clamps to ensure tightness. Design a clamp with holes to ensure that the fluid inlet 101 and the fluid outlet 103 can be connected to other external devices through capillaries.

[0062] The pore size of the porous membrane 2 is set to 8 μm to ensure that the cells are located in the cell culture layer 1 and can carry out normal material exchange with the fluid channel layer 3. The porous membrane 2 uses a PC material, and the cell culture layer 1 and the fluid channel layer 3 use a PMMA material.

[0063] Example 3

[0064] This example provides a device for biomimetic flue gas exposure. The device for biomimetic flue gas exposure includes a microfluidic chip and a flue gas evaluation device.

[0065] The microfluidic chip is assembled from three layers of chip structures, namely, the cell culture layer 1 on the top, the porous membrane 2, and the fluid channel layer 3. On the microfluidic chip, there are provided a reagent fluid inlet 101, a fluid channel 301, a fluid outlet 103, and 4×4 arranged cell culture wells 102. The diameter of the cell culture well 102 is 2.5 mm, the depth is 2.5 mm, and the well spacing is 6 mm. The fluid channel 301 is an S-shaped channel, and the width of the S-shaped channel is 0.4 mm and the depth is 0.2 mm. The fluid inlet 101 and the fluid outlet 103 are located on the cell culture layer 1, aiming to connect the closed fluid channel 301 with the capillary for adding reagents from the outside. The cell culture well 102 is an open channel both above and below in the cell culture layer 1, so that when the microfluidic chip is assembled, the cell culture well 102 can simultaneously participate in the material exchange with the fluid channel 301 through the porous membrane 2 and add cells, culture medium or drugs from the outside.

[0066] Preparation of the microfluidic chip: The upper and lower layer structures of the microfluidic chip, namely, the cell culture layer 1 and the fluid channel layer 3, are prepared by pouring polydimethylsiloxane (PDMS) into a casting mold and thermally baking for shaping. The cured PDMS chip is peeled off, holes are drilled in the upper layer chip, i.e., the cell culture layer 1. After cleaning it with nitrogen, the chip is assembled with a fixture. The assembled chip is placed in a cell culture dish, and 75% ethanol is poured in and vacuum is applied to remove the air inside the microfluidic chip. The microfluidic chip is inspected. The fabricated microfluidic chip is placed under an inverted microscope to observe the details of the microstructures and microchannels to ensure that its microstructures meet the design requirements.

[0067] As described above, after adding cells from the outside to the upper layer cell culture well 102, culture medium, drugs or stimulants can be given to the cell culture well 102 or the fluid channel 301, and the operation method is not uniquely limited. Cells can be cultured and drugs or stimulants can be given to stimulate multiple cell culture wells 102 simultaneously to exert the advantages of high throughput and low cost.

[0068] A micro-injection pump can be externally connected to the fluid inlet 101, and a container for recovering reagents can be externally connected to the fluid outlet 103.

[0069] After successfully culturing lung epithelial cells on the microfluidic chip, the microfluidic chip is placed into the cell exposure chamber 4 of the flue gas evaluation device. The upper and lower parts of the chamber are provided with an exposure chamber air inlet 401 and an exposure chamber air outlet 402. The exposure chamber air inlet 401 is located on the exposure chamber cover 403. A chip mounting platform 404 with pores is arranged in the exposure chamber for mounting the chip. After placing the microfluidic chip, the cell exposure chamber 4 is fixed in a water bath 5. The air inlet and air outlet of the exposure chamber are connected through the water bath air inlet 501 and the water bath air outlet 502. The water bath 5 is provided with a water bath cover 503, and the water bath air inlet 501 is located on the cover. The lower part of the water bath 5 is provided with a water bath air outlet 502, a water bath water inlet 504 and a water bath water outlet 505. Among them, the external length of the cell exposure chamber 4 is 140 mm, the external width is 110 mm, and the external height is 62 mm. The internal length of the cell exposure chamber 4 is 115 mm, the internal width is 85 mm, and the internal height is 42 mm. A mesh plate is arranged inside the cell exposure chamber 4. The thickness of the mesh plate is 4 mm, and the pore diameter is 12 mm. The pore diameters of the exposure chamber air inlet 401 and the exposure chamber air outlet 402 are 16 mm. The external length of the water bath 5 is 235 mm, the external width is 205 mm, and the external height is 215 mm. The internal length of the water bath 5 is 215 mm, the internal width is 185 mm, and the internal height is 195 mm.

[0070] The upper and lower parts of the microfluidic chip body are encapsulated by clamps to ensure tightness. The designed clamp with holes ensures that the fluid inlet 101 and the fluid outlet 103 can be connected to other external devices through capillaries.

[0071] The pore size of the porous membrane 2 is set to 8 μm to ensure that cells are located in the cell culture layer 1 and can carry out normal material exchange with the fluid channel layer 3. The porous membrane 2 uses polycarbonate (PC) material, and the cell culture layer 1 and the fluid channel layer 3 use PDMS material.

[0072] Example 4

[0073] This example provides a method for using the device for biomimetic flue gas exposure of the present invention.

[0074] Culturing lung epithelial cells on the microfluidic chip prepared in Example 1: Before use, the microfluidic chip prepared in Example 1 is placed in a biosafety cabinet and irradiated with ultraviolet light for 30 min, and then washed 3 times with sterile PBS. 2×10 is injected into the cell culture wells of the microfluidic chip 4Lung epithelial cells at a density of 1 cell / 10 μL were placed. After allowing them to settle and adhere, a 1 mL sterile syringe was mounted on a microinjection pump, and DMEM high-glucose medium (containing 10% fetal bovine serum and 1% penicillin) was slowly infused into the fluid channel layer 3 at a rate of 10 μL / min. The microfluidic chip was placed in an incubator at 37 °C and 5% CO₂ for culture. The growth of the cells in the microfluidic chip was observed using a microscope. The cells were cultured until the confluence reached approximately 50 - 70% for simulating the airway-fluid interface.

[0075] Before use, the device for biomimetic smoke exposure described in Example 1 was disinfected by ultraviolet irradiation for 30 min. The water inlet 504 of the water bath was connected to sterile distilled water. The water inlet 504 was opened and the water outlet 505 was closed so that sterile distilled water flowed into the lower part of the water bath 5. The microfluidic chip was fixed in the cell exposure chamber 4. After the exposure chamber was installed, its air inlet 401 and air outlet 402 were respectively connected to the water bath air inlet 501 and air outlet 502 of the smoke stimulation device. The cell exposure chamber 4 was fixed in the water bath 5. After the water bath was installed, cigarette-smoked smoke was introduced into the water bath air inlet 501.

[0076] In summary, the device for biomimetic smoke exposure of the present invention can simulate the three-dimensional microenvironment and dynamic blood flow at the air-liquid interface of the human respiratory tract, can achieve sufficient contact between human lung epithelial cells and air and smoke, can simulate the intensity and frequency of smoke stimulation to the human lungs during smoking, and improve the accuracy of the evaluation of smoke irritation.

[0077] The applicant declares that the present invention uses the above examples to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A device for biological simulation of smoke exposure, characterized in that: The device for biosimulation smoke exposure comprises a microfluidic chip and a smoke evaluation device; the microfluidic chip comprises a cell culture layer (1), a porous membrane (2) and a fluid channel layer (3); the smoke evaluation device comprises a cell exposure box (4) and a water bath (5); The cell culture layer (1) comprises a fluid inlet (101), a cell culture hole (102) and a fluid outlet (103); The fluid channel layer (3) comprises a fluid channel (301); The cell exposure box (4) comprises an exposure box air inlet (401), an exposure box air outlet (402), an exposure box cover (403) and a chip mounting platform (404); The water bath (5) comprises a water bath air inlet (501), a water bath air outlet (502), a water bath cover (503), a water bath water inlet (504) and a water bath water outlet (505).

2. The biological simulation smoke exposure device according to claim 1, characterized in that: The cell culture holes (102) on the cell culture layer (1) are arranged in parallel in the transverse and longitudinal directions; the diameter of the cell culture holes (102) is 1.5-2.5 mm, the depth is 1.5-2.5 mm, and the hole spacing is 4-6 mm.

3. The biological simulation smoke exposure device according to claim 1 or 2, characterized in that: The pore size of the porous membrane (2) is 0.8-8 μm.

4. The device for biological simulation of smoke exposure according to any one of claims 1 to 3, characterized in that: The material of the porous membrane (2) includes polycarbonate.

5. The device for biological simulation of smoke exposure according to any one of claims 1 to 4, characterized in that: The fluid channel (301) is an S-shaped channel, and the width of the S-shaped channel is 0.2-0.4 mm, and the depth is 0.05-0.2 mm.

6. The device for biological simulation of smoke exposure according to any one of claims 1 to 5, characterized in that: The materials of the cell culture layer (1) and the fluid channel layer (3) include polydimethoxysiloxane and / or polymethyl methacrylate.

7. The device for biological simulation of smoke exposure according to any one of claims 1 to 6, characterized in that: The cell exposure box (4) has an external length of 120-140 mm, an external width of 90-110 mm, and an external height of 60-62 mm; the cell exposure box (4) has an internal length of 105-115 mm, an internal width of 75-85 mm, and an internal height of 38-42 mm; a mesh plate is provided inside the cell exposure box (4); the mesh plate has a thickness of 2-4 mm and a pore size of 8-12 mm; Preferably, the apertures of the exposure box air inlet (401) and the exposure box air outlet (402) are 14-16 mm; Preferably, the outer length of the water bath (5) is 225-235 mm, the outer width is 195-205 mm, and the outer height is 205-215 mm, and the inner length of the water bath (5) is 205-215 mm, the inner width is 175-185 mm, and the inner height is 185-195 mm.

8. A method for using the biosimulated smoke exposure device according to any one of claims 1 to 7, characterized in that: The method of use comprises: injecting a fluorescent probe into a cell culture well (102) of the biosimulation smoke exposure device according to any one of claims 1 to 7 to label cells, scanning the cell culture well (102) using a fluorescence microscope, analyzing the type and content of cells labeled with the fluorescent probe by fluorescence intensity, digesting and collecting cells in the cell culture well (102) with trypsin, and extracting RNA for sequencing analysis; The cell culture layer (1), the porous membrane (2) and the fluid channel layer (3) are assembled into a microfluidic chip. The microfluidic chip for culturing lung epithelial cells is placed in a cell exposure box (4). The cell exposure box (4) is placed in a water bath (5). The air inlet (401) and the air outlet (402) of the exposure box are respectively connected to the air inlet (501) and the air outlet (502) of the water bath. The water inlet (504) of the water bath is connected to sterile distilled water. The water inlet (504) is opened and the water outlet (505) is closed to allow sterile distilled water to flow into the bottom of the water bath. Cigarette smoke is introduced into the air inlet (501) of the water bath.

9. Use of the biosimulation smoke exposure device according to any one of claims 1 to 7 in smoke exposure assessment, environmental pollution detection or drug evaluation.

10. The use according to claim 9, characterized in that: The smoke exposure assessment includes a smoke cytotoxicity assessment and a biomarker assessment.

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