A CO2 supply device and method for simulating in vitro CO2 release in vivo

Abstract

The present application relates to the field of in vitro cell experiment, in particular to the real simulation of CO2 supply when culturing cells in vitro, and provides a CO2 supply device and method for simulating in vitro CO2 release in vivo. The CO2 supply device for simulating in vitro CO2 release in vivo comprises a membrane device and a CO2 exhaust device; the membrane device is provided with a hydrophobic gas-permeable membrane, one side of the hydrophobic gas-permeable membrane is a gas communication side, the side is provided with a gas chamber, and the other side of the hydrophobic gas-permeable membrane is a liquid contact side; the CO2 exhaust device has an exhaust port for realizing gas suction; the CO2 supply device for simulating in vitro CO2 release in vivo further comprises a gas flow switching unit, the gas flow switching unit comprises three ports respectively leading to the gas chamber, the exhaust port and a CO2 gas source, and a three-way structure is formed between the three ports, and a valve is arranged on the gas flow path corresponding to each port. The present application can solve the problem that the influence of CO2 on cells is not objective and real in the prior art.

Description

Technical Field

[0001] This invention relates to the field of in vitro cell experiments, and in particular to the realistic simulation of CO2 supply during in vitro cell culture. Background Technology

[0002] During metabolism, cells produce carbon dioxide (CO2), which is transported to the lungs via the bloodstream and expelled through respiration. The form and concentration of CO2 in the blood, as well as the rate at which it is expelled, are crucial for driving respiration and maintaining the body's acid-base balance. Respiration is a vital process for maintaining the balance of oxygen (O2) and CO2 in the body and ensuring metabolism. During this process, the O2 and CO2 content within lung cells constantly changes. During inhalation, air containing a high concentration of O2 and a low concentration of CO2 is inhaled and enters the bloodstream through free diffusion and transmembrane transport via alveolar cells. During exhalation, gas containing a lower concentration of O2 and a higher concentration of CO2 is freely diffused and transported across the membrane to the surface of alveolar cells and expelled from the body.

[0003] Currently, in in vitro cell experiments, gas-liquid interface cell culture methods are widely used in basic and translational medicine research, such as lung drug evaluation, toxicology testing, and disease model construction, because they can simulate the unique gas-liquid exchange interface of the respiratory membrane of lung cells, i.e., the air-blood barrier of the lung. However, existing gas-liquid interface culture and exposure modes do not simulate the high concentration of CO2 released from the blood that the respiratory membrane experiences during exhalation. This results in a significant deviation from simulating the real microenvironment of lung cells in vitro, and further, it will also lead to significant deviations in the study of the mechanisms of CO2-induced diseases such as hyper / hypocapnia and acid / alkalosis.

[0004] An article titled "The Effects of Different Concentrations of CO2 on the Viability of A549 Cells (a Type II Alveolar Epithelial Cell Model)" (Guangxi Medical Journal, February 2015, Vol. 37, No. 2) describes the effects of different concentrations of CO2 on the biological characteristics of A549 cells and explores the lung-protective mechanism of permissive hypercapnia. The article mentions culturing A549 cells in incubators with different concentrations of CO2, but it does not simulate the process of CO2 expulsion from the liquid environment into the gaseous environment through free diffusion and transmembrane transport experienced by lung cells in vivo. Therefore, the pH changes in the culture medium caused by different concentrations of CO2 studied in this article are very different from those in reality.

[0005] Patent document CN209778894U discloses a CO2 incubator for pollutant exposure testing, in which CO2 enters the gaseous environment in gaseous form and is supplied to the incubation unit. Although the technical solution disclosed in this patent document can achieve the supply of CO2, it cannot simulate the discharge process of CO2 from the liquid environment into the gaseous environment through free diffusion and transmembrane transport.

[0006] The aforementioned existing technologies cannot simulate the process by which CO2 is expelled from the liquid environment into the gaseous environment through free diffusion and transmembrane transport. Therefore, they cannot provide a more objective and realistic platform for in vitro studies on the effects of different concentrations of CO2 on cells caused by various reasons (such as excessive CO2 production in vivo, hyperventilation or ventilation impairment, and metabolic acid-base poisoning in vivo). Summary of the Invention

[0007] The purpose of this invention is to provide a CO2 supply device for simulating CO2 release in vitro, solving the problem that existing technologies cannot simulate the discharge process of CO2 from a liquid environment into a gaseous environment via free diffusion and transmembrane transport. Another purpose of this invention is to provide a CO2 supply method for simulating CO2 release in vitro, capable of simulating the release of CO2 from a liquid environment in vivo via free diffusion and transmembrane transport, thus more realistically reflecting the impact of CO2 released in vivo on cells cultured in vitro.

[0008] The present invention adopts the following technical solution:

[0009] A CO2 supply device for simulating CO2 release in vitro includes a membrane device and a CO2 extraction device. The membrane device has a hydrophobic and permeable membrane that allows gas to pass through but prevents liquid from passing through. One side of the hydrophobic and permeable membrane is a gas-connecting side, which has a gas chamber; the other side is a liquid-contacting side, which is used to contact the liquid in the culture unit to supply CO2 to the liquid. The CO2 extraction device has an exhaust port for gas extraction. The CO2 supply device for simulating CO2 release in vitro also includes an airflow switching unit, which has three ports respectively leading to the gas chamber, the exhaust port, and the CO2 gas source. The three ports form a three-way structure, and valves are provided on the airflow paths corresponding to each port.

[0010] Beneficial effects: The hydrophobic and breathable membrane on the membrane device enables full contact between CO2 and the liquid, improving the effective utilization rate and gas supply accuracy of CO2. It also separates CO2 gas from the liquid in the culture unit, avoiding the introduction of factors that may contaminate the culture unit. At the same time, the CO2 extraction device can achieve regular gas extraction through regular operation, thereby forming the required CO2 supply mode. Compared with the existing technology of directly introducing CO2 into the cell culture environment, it can simulate the effect of CO2 on cells by the liquid environment. The process is more realistic and can provide a more objective and realistic platform for studying the effects of different concentrations of CO2 on cells in vivo.

[0011] Furthermore, the hydrophobic and breathable membrane has a liquid contact side for the liquid in the culture unit to enter, and the liquid chamber is provided with a liquid interface.

[0012] Beneficial effects: The liquid chamber and liquid interface facilitate the connection between the membrane device and the culture unit, making it easy to use.

[0013] Furthermore, the airflow switching unit is formed by a switching valve having at least three ports.

[0014] Beneficial effects: The airflow switching unit uses a switching valve, which has a compact structure and facilitates the assembly of the entire device.

[0015] Furthermore, the airflow switching unit is connected to pipelines that lead to the gas chamber, the exhaust cylinder, and the CO2 gas source, respectively, which are the culture unit connection pipeline, the exhaust connection pipeline, and the gas source connection pipeline.

[0016] Beneficial effects: The airflow switching unit can be connected to different pipelines to facilitate connection with the gas chamber, the exhaust cylinder and the CO2 gas source, and it is easy to design the relative position of the airflow switching unit and other components.

[0017] Furthermore, a gas storage chamber is connected to the port on the airflow switching unit that leads to the CO2 gas source, and the gas storage chamber is used to buffer the CO2 supplied by the CO2 gas source.

[0018] Beneficial effects: Buffering the CO2 supply through the gas storage chamber ensures a stable CO2 flow rate, which helps improve simulation accuracy.

[0019] Furthermore, the airflow switching unit also includes a fourth port for connecting to the outside atmosphere, at which a corresponding valve is provided.

[0020] Beneficial effects: With the above structure, the fourth port can connect the gas path to the atmosphere, thereby discharging the gas in the gas path when needed. It has good expandability and can adapt to more usage needs.

[0021] Furthermore, the gas-connected side of the hydrophobic and breathable membrane is connected to an exhaust device for discharging gas that has permeated through the hydrophobic and breathable membrane from the liquid in the culture unit. The exhaust device includes an exhaust cylinder, which contains an exhaust piston, and the exhaust piston is connected to a piston drive device. The exhaust cylinder has two inlet and outlet ports, one of which is for connecting to the membrane device, and the other is for connecting to the atmosphere or a gas collection device. Valves are provided on the airflow paths corresponding to the two inlet and outlet ports.

[0022] Beneficial effects: When the piston drive device of the exhaust device is activated, it can change the gas volume in the exhaust cylinder through the exhaust piston. One inlet and outlet of the exhaust cylinder is used to connect to the membrane device, and the other inlet and outlet is used to connect to the atmosphere or gas collection equipment. By controlling the opening and closing of the valves at each inlet and outlet, the gas entering the gas chamber through the membrane device after the liquid in the culture unit can be discharged, thereby ensuring the stability of the gas environment inside the device.

[0023] Furthermore, it also includes an exhaust switching unit, which includes three ports that lead to the gas chamber, the exhaust cylinder and the CO2 extraction device respectively. The three ports of the exhaust switching unit form a three-way structure. Valves are provided on the airflow paths corresponding to the ports leading to the gas chamber and the CO2 extraction device, or valves are provided on the airflow paths corresponding to all three ports.

[0024] Furthermore: the CO2 extraction device includes an extraction cylinder, an extraction piston is assembled inside the extraction cylinder, and the extraction piston is connected to a piston drive device.

[0025] Beneficial effects: Using a piston design makes it easier to control parameters such as pumping frequency and pumping speed, resulting in more accurate pumping and exhaust waveforms.

[0026] A CO2 supply device for simulating in vivo CO2 release in vitro using any of the above technical solutions, the method comprising the following steps: controlling the corresponding valve of the airflow switching unit to connect the exhaust port of the CO2 extraction device to the CO2 gas source, controlling the operation of the CO2 extraction device to extract gas from the CO2 gas source; then, connecting the exhaust port of the CO2 extraction device to the membrane device, controlling the operation of the CO2 extraction device to push the extracted gas into the gas chamber on the hydrophobic and breathable membrane side, thereby allowing the gas to pass through the hydrophobic and breathable membrane to move to the liquid contact side and enter the liquid in the culture unit; or, controlling the corresponding valve of the airflow switching unit to connect the CO2 gas source to the membrane device, allowing the gas in the CO2 gas source to enter the gas chamber located on the hydrophobic and breathable membrane side, thereby allowing the gas to pass through the hydrophobic and breathable membrane to move to the liquid contact side and enter the liquid in the culture unit.

[0027] Beneficial effects: The hydrophobic and breathable membrane on the membrane device enables full contact between CO2 and the liquid, improving the effective utilization rate and gas supply accuracy of CO2. It also isolates CO2 gas from the liquid in the culture unit, avoiding the introduction of factors that may contaminate the culture unit. At the same time, by controlling the corresponding valves of the airflow switching unit, the CO2 extraction device can achieve regular gas extraction through regular operation, thereby forming the required CO2 supply mode. Compared with the existing technology of directly introducing CO2 into the cell culture environment, it can simulate the effect of carbon dioxide on cells from the liquid environment. The process is more realistic and can provide a more objective and realistic platform for studying the effects of different concentrations of CO2 on cells in vivo.

[0028] Furthermore, the culture unit is equipped with an air inlet and an air outlet. When the air inlet continuously supplies air and the air outlet continuously supplies air, CO2 is continuously supplied to the gas chamber on the membrane device. The flow rate of the supplied CO2 + the flow rate of the air inlet of the culture unit = the flow rate of the air outlet of the culture unit.

[0029] Beneficial effects: The above scheme can ensure the balance of gas intake and exhaust during continuous intake and exhaust of the culture unit.

[0030] Furthermore: The culture unit is equipped with an air inlet and an air outlet. When the air inlet and air outlet are venting air at a set frequency, the CO2 extraction device is controlled to supply CO2 to the gas chamber on the membrane device when venting air from the air outlet. The flow rate of supplied CO2 + the flow rate of the air inlet of the culture unit = the flow rate of the air outlet of the culture unit.

[0031] Beneficial effects: The above scheme can ensure the balance of gas intake and exhaust when the culture unit intakes and exhausts at the set frequency.

[0032] Furthermore, the CO2 extraction device relies on a piston drive device to drive the extraction piston assembled inside the corresponding extraction cylinder to achieve gas extraction.

[0033] Beneficial effects: Using a piston design makes it easier to control parameters such as pumping frequency and pumping speed, resulting in more accurate pumping and exhaust waveforms. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the structure of a CO2 supply device for simulating CO2 release in vitro;

[0035] Figure 2 This is a schematic diagram of the structure of Embodiment 2 of a CO2 supply device used to simulate CO2 release in vitro.

[0036] The names of the components corresponding to the corresponding reference numerals in the figure are as follows: 100, culture unit; 110, external container; 111, liquid external connection port; 120, internal container; 121, air inlet; 122, air outlet; 130, porous membrane; 140, culture medium; 200, membrane device; 210, membrane device cavity; 211, liquid interface; 212, gas interface; 213, gas chamber; 214, liquid chamber; 220, hydrophobic and breathable membrane; 300, exhaust switching unit; 310, left port; 320, right port; 330, lower port; 400, exhaust device; 410, exhaust cylinder; 420. Exhaust piston; 430. Exhaust power unit; 440. Exhaust port; 500. Flow meter; 600. Filter; 610. Filter chamber; 620. Filter membrane; 700. Airflow switching unit; 710. First valve; 720. Second valve; 730. Third valve; 740. Fourth valve; 800. CO2 extraction device; 811. Extraction cylinder; 812. Extraction piston; 813. Extraction power unit; 900. CO2 gas source; 910. Gas storage chamber; 911. Gas inlet; 912. Gas outlet; 920. CO2 cylinder; 930. Check valve / solenoid pressure reducing valve. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, not all embodiments. The components of the embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0038] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0039] It should be noted that, in specific embodiments of the present invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any actual relationship or order between these entities or operations. Furthermore, terms such as "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, the use of phrases such as "comprising a…" to define an element does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0040] In the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linkage" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium, or they can refer to the internal connection of two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0041] In the description of this invention, unless otherwise explicitly specified and limited, the term "provided with" should be interpreted broadly. For example, the object "provided with" can be a part of the body, or it can be separately arranged from the body and connected to the body. This connection can be a detachable connection or a non-detachable connection. Those skilled in the art can understand the specific meaning of the above terms in this invention through specific circumstances.

[0042] The present invention will be further described in detail below with reference to the embodiments.

[0043] Specific embodiment 1 of the CO2 supply device for simulating in vivo CO2 release in vitro in this invention:

[0044] For ease of explanation, we will follow as follows Figure 1 The embodiments are described from a perspective shown below. "Left" and "right" in the following text refer to the perspective shown. Figure 1 The left and right sides are used as references.

[0045] A CO2 supply device for simulating CO2 release in vitro is used to supply... Figure 1The leftmost culture unit 100 supplies CO2. Culture unit 100 simulates the air-blood barrier of the lungs, thereby simulating the process of CO2 being transported through the bloodstream and exhaled through the air-blood barrier of the lungs. The specific structure of culture unit 100 will be described below. The CO2 supply device for simulating CO2 release in vitro includes a membrane device 200, an exhaust device 400, a flow meter 500, a filter 600, an airflow switching unit 700, a CO2 extraction device 800, and a gas storage chamber 910. In use, the gas storage chamber 910 is connected to a CO2 cylinder 920, which serves as the CO2 gas source 900.

[0046] In this embodiment, the culture unit 100 corresponding to the CO2 supply device for simulating CO2 release in vitro includes an outer container 110 containing liquid for forming a liquid layer and an inner container 120 with a porous membrane 130 at the bottom to form an adherent cell layer. The bottom surfaces of the inner container 120 and the outer container 110 are horizontal. The inner container 120 is placed inside the outer container 110, and the sidewall of the inner container 120 is sealed to the outer container 110. The lower right side of the outer container 110 has a liquid connection port 111. The upper end of the inner container 120 has an air inlet 121 and an air outlet 122 to simulate the breathing process of the lungs and realize gas exchange between the culture unit 100 and the outside world. A cell layer is seeded on the porous membrane 130 at the bottom of the inner container 120. The cell layer is divided into a lung cell layer on the upper side of the porous membrane 130 and a vascular endothelial cell layer on the lower side of the porous membrane 130. The lung cell layer, such as A549, Beas-2b, or lung organoids, is in contact with the gas above it; the vascular endothelial cell layer, such as HUVEC or vascular organoids, is in contact with the liquid below it. In this embodiment, the liquid is culture medium 140 containing inflammatory cells, immune cells, and blood cells. In other embodiments, the liquid may not contain cells; additionally, in other embodiments, depending on the experimental purpose, if it is necessary to simulate blood circulation, or if the experiment has a corresponding requirement, the liquid may be allowed to circulate.

[0047] The membrane device 200 is connected to the liquid external connection port 111 on the right side of the culture unit 100. It includes a membrane device cavity 210 and a hydrophobic and breathable membrane 220 that vertically divides the internal space of the membrane device cavity 210 into left and right parts. The hydrophobic and breathable membrane 220 allows gas to pass through but not liquid. Preferably, the hydrophobic and breathable membrane 220 is circular, with its left and right sides being the liquid contact side and the gas communication side, respectively. The hydrophobic and breathable membrane 220 can be made of materials such as polypropylene, poly4-methylpentene, tetrafluoroethylene, or fluoropolymers, which are existing technologies and will not be described in detail here. Correspondingly, the left side of the membrane device cavity 210 is a liquid chamber 214, which has a liquid interface 211 connected to the liquid external connection port 111 of the culture unit 100, allowing liquid from the culture unit 100 to enter the liquid interface 211. The right side of the cavity is a gas chamber 213, which has a gas interface 212 for gas passage. Preferably, the liquid interface 211 is connected to a pH detection unit, which is connected to the control unit when in use.

[0048] Of course, the culture unit 100 in this embodiment is only one application scenario. The CO2 supply device used to simulate CO2 release in vitro can also be connected to other forms of culture unit 100, such as the exposure chamber equipped with Transwell in the prior art, or the organ-on-a-chip equipped with a porous membrane 130 for cell culture. The common feature of these culture units 100 is that they have a porous membrane that can be used for cell culture. The porous membrane serves as a contact layer, and cells can be seeded on both sides, allowing cells to adhere and grow, forming a cell adherent layer. The porous membrane has pores, the diameter of which is smaller than the diameter of the cells. When adherent cells are seeded on the porous membrane, the cells will not leak through the pores. At this time, the cells growing on the membrane form a cell layer. At the same time, the porous membrane can divide the upper and lower layers into two chambers. Depending on the experimental purpose, the upper layer is air, forming an air layer, and the lower layer is a liquid used to wet the porous membrane and cells, forming a liquid layer.

[0049] It should be noted that the CO2 supply device used in this invention to simulate CO2 release in vitro can also be connected to a culture unit using immersion cell culture. In this case, the membrane device 200 can be directly immersed in the culture medium in the culture unit, or the liquid interface 211 of the membrane device 200 can be connected to the culture medium container.

[0050] The exhaust device 400 includes an exhaust cylinder 410, an exhaust piston 420, and an exhaust power unit 430. The exhaust cylinder 410 is arranged vertically, and the exhaust power unit 430, acting as a piston drive, drives the exhaust piston 420 to move vertically within the exhaust cylinder 410. Alternatively, in other embodiments, the exhaust cylinder 410 can be arranged horizontally or at an angle, depending on the application requirements. The volume of the exhaust cylinder 410 can be selected as needed. The pull rod on the exhaust piston 420 can be a straight rod or a bent rod, working in conjunction with the exhaust power unit 430 to push and pull the exhaust piston 420, causing the exhaust cylinder 410 to intake and exhaust air. The exhaust cylinder 410 is made of materials such as transparent glass or hard plastic. The exhaust power unit 430 uses a device that converts rotation into telescopic motion, driven by a motor, and can drive the exhaust piston 420 at a set speed and frequency. Such a device can be implemented without creative effort by those skilled in the art.

[0051] The CO2 supply device for simulating CO2 release in vitro also includes an exhaust switching unit 300, which includes a three-way valve comprising a three-way chamber and a solenoid valve. The three-way chamber has three ports: a left port 310, a right port 320, and a lower port 330. The left port 310 is connected to the gas interface 212 of the membrane device 200, providing access to the membrane device 200; the right port 320 is connected to the lower end of the flow meter 500, providing access to the airflow switching unit 700; the exhaust cylinder 410 has two inlet and outlet ports at its top, one of which is connected to the lower port 330 of the exhaust switching unit 300, providing access to the membrane device 200, and the other providing access to the atmosphere. Valves are installed along the airflow paths corresponding to both inlet and outlet ports. Each port of the exhaust switching unit 300 is equipped with a solenoid valve, which is connected to the corresponding control unit during use. For polluted gases, the inlet and outlet ports for accessing the atmosphere can be connected to a gas collection device. In other embodiments, the exhaust switching unit 300 may be part of the exhaust device 400.

[0052] Flow meter 500 measures the flow rate of CO2 passing through it, and the material in contact with the gas is sterilizable. The upper end of flow meter 500 is connected to the lower end of filter 600. This is a bidirectional flow meter and can be installed in different locations as needed.

[0053] The filter 600 consists of a filter chamber 610 and a filter membrane 620. The filter chamber 610 can be made of stable metal, plastic, etc., and is sterilizable. Both the upper and lower ends of the filter chamber 610 are threaded, ensuring good airtightness when connected to corresponding gas pipelines. The filter membrane 620 is a sterilizing membrane, located at the cross-section of the filter chamber 610, with its edges sealed to the filter chamber 610, providing sterilization and filtration for passing CO2. The filter 600 can also be installed at the CO2 gas source generator end, both filtering the gas and reducing the filter's impact on flow rate.

[0054] The airflow switching unit 700 is a four-way switching valve, including a four-way cavity. The four-way cavity has four ports leading to the gas chamber 213, the exhaust cylinder 811, the CO2 gas source 900, and the outside atmosphere, respectively. These four ports form a four-way structure, and each port's corresponding airflow path is equipped with a solenoid valve. The four-way cavity is a four-way pipe-accessible cavity, and its shape can be arbitrary. Solenoid valves are located on each directional port, namely the first valve 710, the second valve 720, the third valve 730, and the fourth valve 740. The control unit controls the opening and closing of each valve and the degree of opening and closing to precisely control the airflow direction and flow rate in the pipeline. The four-way cavity is made of a malleable, robust material with a smooth inner surface and chemically stable properties that are not prone to chemical reactions; typically, it is made of glass, hard plastic, or stainless steel.

[0055] The structure of the CO2 extraction device 800 is similar to that of the exhaust device 400, including an extraction cylinder 811, an extraction piston 812, and an extraction power unit 813 for driving the piston to extend and retract left and right according to a set frequency and speed. The extraction power unit 813 constitutes a piston drive device. The extraction cylinder 811 has an inlet and outlet on its left side for extracting gas from the CO2 gas source 900 and supplying gas to the culture unit 100.

[0056] A CO2 gas source 900 is connected to a gas storage chamber 910, which includes a CO2 cylinder 920 and a check valve 930. The gas storage chamber 910 buffers the gas supplied by the CO2 cylinder 920. The check valve 930 at the upper end of the CO2 cylinder 920 prevents gas in the gas storage chamber 910 from flowing back into the CO2 cylinder 920. The gas storage chamber 910 has a gas inlet 911 and a gas outlet 912, and houses a CO2 concentration detector (not shown in the figure). Electromagnetic valves are installed at the gas inlet 911 and the gas outlet 912. The check valve 930 is connected to the gas inlet 911 of the gas storage chamber 910 via an outlet pipe, and the gas outlet 912 of the gas storage chamber 910 is connected to the lower port of a four-way switching valve. Of course, the electromagnetic valves at the gas inlet 911 and the gas outlet 912, along with all the valves mentioned above, are connected to a control unit. Depending on the requirements, CO2 cylinder 920 can be a standard gas cylinder with a calibrated CO2 gas concentration, or it can be a cylinder containing pure CO2, which is mixed with N2 or synthetic air at the target concentration when in use.

[0057] The connecting pipes between the corresponding parts of the CO2 supply device used to simulate CO2 release in vitro are sterilizable glass tubes, plastic tubes, soft rubber tubes, etc.

[0058] The usage process of the CO2 supply device used in this embodiment to simulate CO2 release in vitro is as follows:

[0059] Under normal circumstances, the left port 310 and right port 320 on the exhaust switching unit 300 corresponding to the exhaust device 400 remain open, while the lower port 330 can remain closed or open. The solenoid valves at the two exhaust ports on the left and right sides of the inlet and outlet at the top of the exhaust cylinder 410 are closed. When it is necessary to simulate CO2 release externally, the check valve 930 on the CO2 gas source 900 is opened, allowing the CO2 gas in the CO2 cylinder 920 to enter the storage chamber 910 after depressurization. Then, according to the experimental requirements, the control unit controls the second valve 720 and the third valve 730 on the airflow switching unit 700 to open, and the first valve 710 and the fourth valve 740 to close, connecting the extraction cylinder 811 to the storage chamber 910. The extraction power unit 813 drives the extraction piston 812 to move to the right, drawing the gas in the storage chamber 910 into the extraction cylinder 811. During this period, the CO2 cylinder 920 continuously supplies gas to the storage chamber 910. Then, the first valve 710 and the third valve 730 on the airflow switching unit 700 of the control unit are closed, and the second valve 720 and the fourth valve 740 are opened. The pumping power unit 813 drives the pumping piston 812 to move to the left, supplying the CO2 in the pumping cylinder 811 into the liquid contained in the external container 110 of the cell culture device through the filter 600, the flow meter 500, and the membrane device 200. Part of the CO2 reacts chemically with the liquid in the culture unit 100 to generate extremely unstable H2CO3, which affects the pH value of the overall system. Part of the CO2 diffuses freely to the cell surface and is transported across the membrane to the cell adherent layer above the porous membrane 130, completing the exhalation process of CO2 freely diffusing from the blood and being transported across the membrane to the surface of the alveolar cells. The CO2 exhaled to the cell surface is pumped out by the pump connected to the air outlet 122 of the culture unit 100 or overflows freely.

[0060] According to experimental requirements, if CO2 needs to be continuously introduced into the liquid in the culture unit 100, the first valve 710 and the second valve 720 can be closed, and the third valve 730 and the fourth valve 740 can be opened. The gas output from the CO2 gas source 900 is continuously supplied to the membrane device 200 through the gas storage chamber 910.

[0061] If some of the gas entering through the air inlet 121 of the culture unit 100 passes through the cell adhesion layer and enters the liquid, the free gas can enter the gas chamber 213 through the membrane device 200. At this time, the control unit controls the right port 320 of the exhaust switching unit 300 to close and the left port 310 and the lower port 330 to open. The solenoid valves at the air inlets and outlets of the exhaust cylinder 410 leading to the exhaust switching unit 300 open and the air inlets and outlets leading to the atmosphere close. The exhaust power unit 430 is activated to draw the gas entering from the culture unit 100 through the membrane device 200 into the exhaust cylinder 410. Then, the solenoid valves at the air inlets and outlets of the exhaust cylinder 410 leading to the exhaust switching unit 300 close, the air inlets and outlets leading to the atmosphere open, the lower port 330 closes, and the exhaust power unit 430 is activated to discharge the gas in the exhaust cylinder 410 to maintain the gas pressure balance in the culture unit 100.

[0062] Example 2 of the CO2 supply device for simulating in vivo CO2 release in vitro according to the present invention:

[0063] The difference between this embodiment and Embodiment 1 is that in Embodiment 1, the liquid contact side of the hydrophobic and breathable membrane 220 is provided with a liquid chamber 214 for the liquid in the culture unit 100 to enter, and the liquid chamber 214 is provided with a liquid interface 211. In this embodiment, however, the liquid contact side of the hydrophobic and breathable membrane 220 is directly exposed, and there is no liquid chamber 214 for the liquid in the culture unit 100 to enter. When in use, the membrane device 200 extends directly into the liquid in the culture unit 100.

[0064] Example 3 of the present invention: A CO2 supply device for simulating in vivo CO2 release in vitro:

[0065] The difference between this embodiment and Embodiment 1 is as follows: In Embodiment 1, the airflow switching unit 700 includes four ports, one of which is connected to the atmosphere. The filter 600 is a bidirectional filter 600, which can draw gas from the culture unit 100 into the gas chamber 213 via the membrane device 200 through the CO2 extraction device 800 into the extraction cylinder 811, and then discharge it through the port on the airflow switching unit 700 connected to the atmosphere. In this embodiment, the airflow switching unit 700 only includes three ports, which are respectively connected to the gas chamber 213, the extraction cylinder 811, and the CO2 gas source 900. In this case, the filter 600 can be a unidirectional filter 600, used only to filter the CO2 gas flowing to the culture unit 100.

[0066] Example 4 of the present invention: A CO2 supply device for simulating in vivo CO2 release in vitro:

[0067] The difference between this embodiment and Embodiment 1 is that in Embodiment 1, the airflow switching unit 700 is formed by a switching valve with at least three ports. In this embodiment, the airflow switching unit 700 includes a three-way connector, which is connected to the gas chamber 213, the exhaust cylinder 811, and the CO2 gas source 900 through pipelines, and valves are installed on each pipeline.

[0068] Example 5 of the present invention: A CO2 supply device for simulating in vivo CO2 release in vitro:

[0069] The difference between this embodiment and Embodiment 1 is that in Embodiment 1, the exhaust switching unit 300 corresponding to the exhaust device 400 includes a three-way valve and two inlet / outlet ports located at the top of the exhaust cylinder 410, with solenoid valves installed at each port of the three-way valve and at the two inlet / outlet ports. In this embodiment, the exhaust switching unit 300 corresponding to the exhaust device 400 only includes two inlet / outlet ports located at the top of the exhaust cylinder 410, respectively leading to the gas chamber 213 and the atmosphere, with solenoid valves installed at both inlet / outlet ports. Of course, in other embodiments, since valves are already installed at the inlet / outlet ports at the top of the exhaust cylinder 410 leading to the gas chamber 213, valves may not be installed at the lower port of the exhaust switching unit 300; or, valves may be installed at the lower port of the exhaust switching unit 300, while valves may not be installed at the inlet / outlet ports at the top of the exhaust cylinder 410 leading to the gas chamber 213.

[0070] Example 6 of the present invention: A CO2 supply device for simulating in vivo CO2 release in vitro:

[0071] The difference between this embodiment and Embodiment 1 is that in Embodiment 1, the CO2 gas source 900 is connected to the airflow switching unit 700 through the gas storage chamber 910 and the gas source connection pipeline. In this embodiment, however, the interface of the gas storage chamber 910 is directly connected to the airflow switching unit 700. Alternatively, in other embodiments, the gas storage chamber 910 can be omitted, and the CO2 gas source 900 can be directly connected to the airflow switching unit 700 or connected to the airflow switching unit 700 through the gas source connection pipeline.

[0072] Example 7 of the present invention: A CO2 supply device for simulating in vivo CO2 release in vitro:

[0073] The difference between this embodiment and Embodiment 1 is that in Embodiment 1, the CO2 extraction device is in the form of a piston, including an extraction cylinder 811, an extraction piston 812, and a piston drive device. In this embodiment, the CO2 extraction device uses an extraction pump to draw gas into the corresponding gas chamber and discharge it from the gas chamber.

[0074] Example 1 of the CO2 supply method for simulating in vivo CO2 release in vitro in this invention:

[0075] This method uses the apparatus described in Example 1 of the CO2 supply device for simulating in vivo CO2 release in vitro, and is a method for simulating the CO2 release process experienced by the lung air-blood barrier in vitro. It includes the following steps:

[0076] 1. In a sterile environment, connect the sterile liquid interface 211 on the liquid contact side of the hydrophobic and breathable membrane 220 to the liquid external connection port 111 of the sterile culture unit 100.

[0077] 2. In vitro simulation of the lung air-blood barrier: Lung cells, such as A549, Beas-2b, or lung organoids, are seeded above the porous membrane 130 of culture unit 100, and vascular endothelial cells, such as HUVEC or vascular organoids, are seeded below. The vascular endothelial cells are in contact with the liquid, which is a culture medium 140 containing inflammatory, immune, and blood cells, such as macrophages and blood cells.

[0078] 3. In vitro simulation of CO2 release across the lung air-blood barrier under normal physical conditions:

[0079] 1) The air inlet 121 and air outlet 122 of the culture unit 100 are connected to the corresponding air pumps. The gas inlet and outlet parameters of the air inlet 121 and air outlet of the culture unit 100 are set so that the air can be continuously inlet or inlet and outlet can be inlet and outlet according to the respiratory rhythm.

[0080] 2) Set the control unit parameters for the CO2 supply device used to simulate CO2 release in vitro, including expiratory frequency, expiratory time, CO2 concentration, and flow rate. If the in vitro simulation of the lung air-blood barrier is normal, and the expiratory frequency and expiratory time are normal parameters, due to the different gas layer volumes and liquid layers in different culture units 100, to ensure the gas pressure environment of the exhaled CO2 culture unit 100 and the pH range of the liquid layer (maintaining the normal lung air-blood barrier pH value, such as around 7.2), the CO2 flow rate and concentration parameters are as follows:

[0081] a. When the culture unit 100 is continuously inlet and outlet, the CO2 concentration is 4%-5% when CO2 is introduced; the flow rate of supplied CO2 + the flow rate of the inlet 121 of the culture unit 100 = the flow rate of the outlet 122 of the culture unit 100; when CO2 is not introduced, the continuous gas inlet flow rate is the same as the continuous gas outlet flow rate.

[0082] b. When the culture unit 100 inhales and exhales according to the respiratory rhythm, the frequency of CO2 introduction is consistent with the exhalation frequency of the culture unit 100, and the concentration of CO2 introduced is 4%-5%; the flow rate of supplied CO2 + the flow rate of the inlet 121 of the culture unit 100 = the flow rate of the outlet 122 of the culture unit 100. The supply CO2 flow rate is usually less than 1.6 times the gas layer volume of the culture unit 100.

[0083] Of course, in other embodiments, the culture unit 100 may also operate at other set frequencies as needed, and does not necessarily have to follow the breathing rhythm corresponding to the normal breathing process of the human body for inhalation and exhalation.

[0084] Example 2 of the CO2 supply method for simulating in vivo CO2 release in vitro in this invention:

[0085] The difference between this embodiment and Embodiment 1 is that Embodiment 1 was used to simulate the CO2 release process across the air-blood barrier under normal physical conditions in vitro, while this embodiment is used to simulate the CO2 release process across the air-blood barrier under the physical conditions of metabolic acidosis in vitro, thereby obtaining in vitro models with different clinical characteristics:

[0086] 1) The air inlet 121 and air outlet 122 of the culture unit 100 are connected to the corresponding air pumps. The gas inlet and outlet parameters of the air inlet 121 and air outlet of the culture unit 100 are set so that the air can be continuously inlet or inlet and outlet can be inlet and outlet according to the respiratory rhythm.

[0087] 2) Configure the control unit parameters for the CO2 supply device used to simulate CO2 release in vitro, including exhalation frequency, exhalation time, CO2 concentration, and flow rate. The parameters for CO2 flow rate and concentration are as follows:

[0088] a. When the culture unit 100 is continuously inlet and outlet, the concentration of CO2 introduced is greater than 5%; the flow rate of supplied CO2 + the flow rate of inlet 121 of culture unit 100 = the flow rate of outlet 122 of culture unit 100; when CO2 is not introduced, the continuous gas inlet flow rate is the same as the continuous gas outlet flow rate.

[0089] b. When the culture unit 100 inhales and exhales according to the respiratory rhythm, the frequency of CO2 introduction is consistent with the exhalation frequency of the culture unit 100, and the concentration of introduced CO2 is greater than 5%; the flow rate of supplied CO2 + the flow rate of the inlet 121 of the culture unit 100 = the flow rate of the outlet 122 of the culture unit 100. The supply CO2 flow rate is usually less than 1.6 times the gas layer volume of the culture unit 100.

[0090] Example 3 of the present invention: A CO2 supply method for simulating in vivo CO2 release in vitro:

[0091] The difference between this embodiment and Embodiment 1 is that, in Embodiment 1, the culture unit 100 corresponding to the CO2 supply device for simulating in vivo CO2 release in vitro includes an outer container 110 and an inner container 120 with a porous membrane 130 at the bottom to form an adherent cell layer; while in this embodiment, as... Figure 2 As shown, the culture unit 100 does not have an air inlet 121 and an air outlet 122.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the scope of protection of the present invention.

Claims

1. CO2 supply device for in vitro simulation of in vivo CO2 release, characterized in that The device comprises a membrane device (200) and a CO2 extraction device (800). The membrane device (200) is provided with a hydrophobic gas-permeable membrane (220) for allowing gas to pass through but preventing liquid from passing through. One side of the hydrophobic gas-permeable membrane (220) is a gas communication side, which is provided with a gas chamber (213). The other side of the hydrophobic gas-permeable membrane (220) is a liquid contact side, which is used to contact liquid in a culture unit (100) to supply CO2 to the liquid. The CO2 extraction device (800) has an extraction port for realizing gas extraction. A CO2 supply device for simulating in-vivo CO2 release in-vitro further comprises a gas flow switching unit (700). The gas flow switching unit (700) comprises three ports respectively connected to the gas chamber (213), the extraction port and a CO2 source (900), and forms a three-way structure among the three ports. Valves are arranged on the gas flow paths corresponding to the ports. The gas communication side of the hydrophobic gas-permeable membrane (220) is connected with an exhaust device (400) for exhausting gas that has passed through the hydrophobic gas-permeable membrane (220) from the liquid in the culture unit (100). The device further comprises an exhaust switching unit (300). The exhaust switching unit (300) comprises three ports respectively connected to the gas chamber (213), the exhaust device (400) and the CO2 extraction device (800), and forms a three-way structure among the three ports. Valves are arranged on the gas flow paths corresponding to the ports connected to the gas chamber and the CO2 extraction device (800), or valves are arranged on the gas flow paths corresponding to the three ports. A gas storage chamber (910) is connected to the port of the gas flow switching unit (700) for connecting to the CO2 source (900). The gas storage chamber (910) is used to buffer CO2 supplied by the CO2 source (900).

2. CO2 supply device for simulating in vitro the release of CO2 in vivo according to claim 1, characterized in that The liquid contact side of the hydrophobic gas-permeable membrane (220) is provided with a liquid chamber for allowing liquid in the culture unit (100) to enter. A liquid interface (211) is arranged on the liquid chamber.

3. CO2 supply device for simulating in vitro the release of CO2 in vivo according to claim 1 or 2, characterized in that The gas flow switching unit (700) is formed by a switching valve having at least three ports.

4. CO2 supply device for simulating in vitro the release of CO2 in vivo according to claim 1 or 2, characterized in that The gas flow switching unit (700) is connected with pipelines respectively for connecting to the gas chamber (213), the extraction cylinder (811) and the CO2 source (900), i.e. a culture unit connecting pipeline, an extraction connecting pipeline and a gas source connecting pipeline.

5. CO2 supply device for simulating in vitro the release of CO2 in vivo according to claim 1 or 2, characterized in that The gas flow switching unit (700) further comprises a fourth port for connecting to the outside atmosphere, and a corresponding valve is arranged at the port.

6. CO2 supply device for simulating in vitro the release of CO2 in vivo according to claim 1 or 2, characterized in that The exhaust device (400) comprises an exhaust cylinder (410) connected to the exhaust switching unit. An exhaust piston (420) is arranged in the exhaust cylinder (410). The exhaust piston (420) is connected with a piston driving device. The exhaust cylinder (410) has two gas inlet and outlet ports. One of the two gas inlet and outlet ports is used to connect to the membrane device (200), and the other is used to connect to the atmosphere or a gas collection device. Valves are arranged on the gas flow paths corresponding to the two gas inlet and outlet ports.

7. CO2 supply device for simulating in vitro the release of CO2 in vivo according to claim 1 or 2, characterized in that The CO2 extraction device (800) comprises an extraction cylinder (811) in which an extraction piston (812) is assembled, and the extraction piston (812) is connected with a piston driving device.

8. CO2 supply method for simulating in vitro the CO2 release in vivo using a CO2 supply device according to any one of claims 1 to 7, characterized in that, The method comprises the following steps: controlling the corresponding valves of the gas flow switching unit (700) to make the extraction air outlet of the CO2 extraction device (800) open to the CO2 gas source (900), and controlling the CO2 extraction device (800) to extract the gas in the CO2 gas source (900); then, making the extraction air outlet of the CO2 extraction device (800) open to the membrane device (200), and controlling the CO2 extraction device (800) to push the extracted gas into the gas chamber (213) on the liquid contact side of the hydrophobic gas-permeable membrane (220), and then make the gas move to the liquid contact side through the hydrophobic gas-permeable membrane (220) and enter the liquid in the culture unit (100); or, controlling the corresponding valves of the gas flow switching unit (700) to make the CO2 gas source (900) open to the membrane device (200), and making the gas in the CO2 gas source (900) enter the gas chamber (213) on the liquid contact side of the hydrophobic gas-permeable membrane (220), and then make the gas move to the liquid contact side through the hydrophobic gas-permeable membrane (220) and enter the liquid in the culture unit (100).

9. The CO2 supply method for simulating in vitro CO2 release in vivo according to claim 8, characterized by, The culture unit (100) is provided with an air inlet (121) and an air outlet (122), when the air inlet (121) continuously supplies air and the air outlet (122) continuously discharges air, the CO2 in the gas chamber on the membrane device (200) is continuously supplied, and the flow of the supplied CO2 + the flow of the air inlet (121) of the culture unit (100) = the flow of the air outlet (122) of the culture unit (100).

10. The CO2 supply method for simulating in vitro CO2 release in vivo according to claim 8, characterized by, The culture unit (100) is provided with an air inlet (121) and an air outlet (122), when the air inlet (121) and the air outlet (122) respectively supply air and discharge air according to a set frequency, the CO2 in the gas chamber on the membrane device (200) is supplied by the CO2 extraction device (800) when the air outlet (122) discharges air, and the flow of the supplied CO2 + the flow of the air inlet (121) of the culture unit (100) = the flow of the air outlet (122) of the culture unit (100).

11. The CO2 supply method for simulating in vitro CO2 release in vivo according to claim 8, characterized by, The CO2 extraction device is driven by the piston driving device to drive the extraction piston assembled in the corresponding extraction cylinder to extract the gas.

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