A cell culture device and a method for enriching cell culture medium with hydrogen

Abstract

The present invention discloses a cell culture device, including a dish cover and a dish body arranged up and down, a connecting sleeve is arranged between the dish cover and the dish body, the outer wall of the connecting sleeve is covered with a fixed disc, the inner wall of the top cover of the dish cover is fixed with a support sheet, a placement cavity a is formed inside the dish body, a filter membrane is arranged inside the placement cavity a, a placement cavity b is formed inside the connecting sleeve, and the dish cover, the connecting sleeve, the dish body and the fixed disc are all made of transparent polystyrene material. The present invention also specifically discloses a method for hydrogen enrichment of the culture medium of the above-mentioned cell culture device, and the hydrogen-enriched culture medium can not only meet the research needs of the above-mentioned scientific researchers for hydrogen-enriched culture medium, but also can be widely promoted because of its simple preparation process, low cost, and non-toxic and harmless use.

Description

Technical Field

[0001] The invention belongs to the field of cell culture, and in particular relates to a cell culture device and a method for enriching cell culture medium with hydrogen. Background Art

[0002] In in vitro cell culture experiments, various cell culture media such as RPMI-1640 Medium, Minimum Essential Medium (MEM), DMEM-high glucose (standard), DMEM-low glucose (standard), etc. are often used. However, during the cell culture process, cells will inevitably produce a variety of oxygen free radicals (ROS) through metabolism, such as hydroxyl free radicals, which will mainly cause lipid peroxidation in the body and damage cells, and can cause damage to the biological membrane system and intracellular oxidative phosphorylation disorders. It will also react with any substance in the body, such as sugars, amino acids, phospholipids, nucleic acids, organic acids, etc., especially with purines and pyrimidines in DNA, leading to cell death or mutation.

[0003] As a new type of antioxidant, hydrogen (H2) has received extensive attention from the medical community at home and abroad in recent years. It can be used as a medical gas. Hydrogen can specifically inhibit harmful ROS, maintain metabolic redox reactions and other weaker ROS, such as hydrogen peroxide and nitric oxide, and can protect cells by regulating oxidative stress, inflammation, and apoptosis. With the development of hydrogen molecular medicine, the antioxidant, anti-inflammatory, and anti-apoptotic biological effects of hydrogen and hydrogen-rich liquids have been confirmed in animals by many research units around the world. However, there is no clear report on the specific related mechanism so far, and a large number of in vitro experiments are needed to explore and verify it.

[0004] Studies have found that the dissolved content of hydrogen in the liquid must be greater than a certain concentration to play an effective biological role. Lu Ruiting and others found that hydrogen-rich water has a protective effect on the damage caused by oxidative stress in mouse astrocytes. RL, YL, xueWDJZwzbjjy. Protective effect of hydrogen-rich water on oxidative stress cell model and the impact of the phosphatidylinositol3kinase / protein kinase B pathway Ryoko Asada, Yasukazu Saitoh and others showed that bathing in hydrogen-rich water with a hydrogen content of 300 ppb can improve visceral fat and spots on the skin;

[0005] A paper published by Tianjin Medical University: Protective effect of hydrogen-containing culture medium on lipopolysaccharide (LPS)-induced human umbilical vein endothelial cell damage. Han Huanzhi et al. also showed that hydrogen-containing culture medium with a hydrogen content of 300 ppb has a protective effect on lipopolysaccharide (LPS)-induced human umbilical vein endothelial cell damage, and as the ppb content increases, the protective effect of hydrogen on cells will also increase.

[0006] Hydrogen molecules are more active due to their small molecular weight. There are currently two methods for preparing commercial hydrogen-rich liquids on the market: one is to fill the solution with hydrogen under high pressure and low temperature conditions to increase the solubility of hydrogen; the other is to ionize water. Regardless of which method is used, the prepared hydrogen-rich liquid needs to be stored in an aluminum can. Through our research, we found that electrolysis of the culture medium will destroy the original nutrients in the culture medium, so hydrogen-rich culture medium cannot be prepared by electrolysis. If you want to prepare a hydrogen-rich culture medium, you can only prepare it by filling it with hydrogen at low temperature. The prepared culture medium can only contain a certain amount of hydrogen in a plastic culture bottle for a very short time, and the hydrogen content that can volatilize biological effects cannot be guaranteed, and it cannot meet the cell culture requirements for exploring its related mechanisms.

[0007] In addition, most of the current methods for obtaining oxygen are through chemical and electrolytic methods. However, the chemical method will produce soluble substances that may affect the composition of the culture medium and thus affect the experiment. Our experiments found that the electrolytic method is not feasible either. Electrolyzing the cell culture medium will not only destroy the original nutrients in the culture medium but also fail to produce oxygen, so it is impossible to obtain a high-oxygen culture medium.

[0008] In summary, since some diseases show symptoms of hypoxia and high concentrations of free radicals, but there is a lack of research methods for these diseases, we have invented a new cell culture device. Through this device and technology, liquid culture medium and high-oxygen and hydrogen-rich culture medium can be obtained to complete various cell experiments, and ultimately provide solutions for the treatment of these diseases, as well as provide a basis for studying the biological activity of oxygen and hydrogen. Summary of the invention

[0009] The first object of the present invention is to provide a cell culture device, which combines hydrogenated coral calcium with a liquid culture medium to achieve the purpose of enriching the liquid culture medium with hydrogen to form a hydrogen-rich culture medium, and can also enrich the high oxygen culture medium with hydrogen to form a hydrogen-rich culture medium.

[0010] Another object of the present invention is to provide a method for enriching a cell culture medium with hydrogen.

[0011] The first technical solution adopted by the present invention is: a cell culture device, including a dish cover and a dish body arranged up and down, a connecting sleeve is provided between the dish cover and the dish body, the outer wall of the connecting sleeve is covered with a fixed disc, the inner wall of the top cover of the dish cover is fixed with a support sheet, the interior of the dish body forms a placement cavity a, the placement cavity a is provided with a filter membrane, the interior of the connecting sleeve forms a placement cavity b, and the dish cover, the connecting sleeve, the dish body and the fixed disc are all made of transparent polystyrene material.

[0012] The first technical solution adopted by the present invention is also characterized in that:

[0013] The dish cover and the dish body are both cylindrical, and the connecting sleeve is annular.

[0014] The inner diameter of the dish cover is larger than the outer diameter of the connecting sleeve, and the outer diameter of the dish cover is not larger than the outer diameter of the fixed disc.

[0015] The support sheet is located just above the upper edge of the connecting sleeve, and the distance from the support sheet to the upper edge of the connecting sleeve is smaller than the distance from the lower edge of the dish cover to the fixed disc.

[0016] Adhesive is arranged at the connection between the connecting sleeve and the dish body.

[0017] An upper thread is arranged on the outer side of the bottom of the connecting sleeve, the upper thread is located at the bottom of the fixed disc, and a lower thread is arranged on the inner side wall of the dish body, and the upper thread and the lower thread match each other.

[0018] The filter membrane is circular, and the diameter of the filter membrane is larger than the inner diameter of the placement cavity a.

[0019] The second technical solution adopted by the present invention is: a method for enriching the culture medium with hydrogen, which is specifically implemented according to the following steps:

[0020] The liquid culture medium is placed in the placement chamber b of the connecting sleeve, and hydrogenated coral calcium is added into the placement chamber a. When the water molecules in the liquid culture medium contact with the hydrogenated coral calcium through the filter membrane to generate hydrogen and merge into the liquid culture medium, a hydrogen-rich culture medium is obtained.

[0021] The second technical solution adopted by the present invention is also characterized in that:

[0022] The liquid culture medium can also be replaced by a hyperoxic culture medium, which is prepared according to the following method: the sterile culture medium is poured into a plastic sealed bottle, and the bottle is placed in a sterile operating room with a medical oxygen generator for oxygenation to obtain the hyperoxic culture medium.

[0023] The beneficial effects of the present invention are as follows: the present invention provides a method for preparing a hydrogen-rich culture medium and a related cell culture device, which can be used by laboratory staff in conjunction with various conventional liquid culture media such as RPMI-1640 Medium, Minimum Essential Medium (MEM), DMEM-high sugar (standard type), and DMEM-low sugar (standard type). The hydrogen-rich culture medium can not only meet the research needs of the above-mentioned researchers for liquid culture media and high-oxygen hydrogen-rich culture media, but also can be widely promoted because of its simple preparation process, low cost, and non-toxic and harmless use. This study provides a platform for relevant scientific researchers to conduct in vitro research, which will have a significant impact and breakthrough in the research work in the field of hydrogen molecular medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a structural schematic diagram of a cell culture device of the present invention;

[0025] Figure 2 A top view of a cell culture device of the present invention;

[0026] Figure 3 It is a structural schematic diagram of a connecting sleeve of a cell culture device of the present invention;

[0027] Figure 4 It is a structural schematic diagram of a cell culture device vessel of the present invention;

[0028] Figure 5 It is a schematic structural diagram of a dish cover of a cell culture device of the present invention;

[0029] Figure 6 It is a line graph of the hydrogen content released in water by hydrogenated coral calcium with different concentrations of the present invention;

[0030] Figure 7 It is a line graph of the hydrogen content of the culture medium of 7.5 g / L hydrogenated coral calcium of the present invention;

[0031] Figure 8 It is a line graph of the hydrogen content of hydrogen-rich water of the present invention;

[0032] Fig. 9 It is a line graph of hydrogen content of 7.5 g / L hydrogenated coral calcium in culture medium, in water, and in hydrogen-rich water of the present invention;

[0033] Fig.10 A comparison of dissolved oxygen content between the oxygen-enriched culture medium of the present invention and other high-oxygen solutions;

[0034] Fig.11 Graph showing oxygen content of the oxygen-enriched culture medium of the present invention in a culture dish and a culture bottle.

[0035] In the figure: 1. dish cover, 2. connecting sleeve, 3. filter membrane, 4. dish body, 5. fixed disk, 6. upper thread, 7. support sheet, 8. lower thread, 9. placement chamber a, 10. placement chamber b. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] like Figure 1 and Figure 2 , which are a schematic diagram and a top view of a cell culture device of the present invention, respectively, including a dish cover 1 and a dish body 4 arranged up and down, a connecting sleeve 2 is provided between the dish cover 1 and the dish body 4, and a fixed disc 5 is provided on the outer wall of the connecting sleeve 2. The structure of the connecting sleeve and the fixed disc is as shown in FIG. Figure 3 As shown, the outer side of the bottom of the connecting sleeve 2 is provided with an upper thread 6, and the upper thread 6 is located at the bottom of the fixed disc 5. Figure 4 As shown, the inner wall of the dish body 4 is provided with a lower thread 8, the upper thread 6 matches the lower thread 8, a placement cavity a9 is formed inside the dish body 4, the filter membrane 3 is laid flat inside the placement cavity a9, and a placement cavity b10 is formed inside the connecting sleeve 2.

[0038] The dish cover 1 and the dish body 4 are both cylindrical, the filter membrane 3 is circular, and the diameter of the filter membrane 3 is larger than the inner diameter of the placement cavity a9.

[0039] The dish cover 1, the connecting sleeve 2, the dish body 4 and the fixed disc 5 are all made of transparent polystyrene (PS) material, which is transparent and hard (mirror surface) and convenient for observation under a microscope. The culture dish cover 1, the connecting sleeve 2 and the dish body 4 are sealed and connected to ensure that hydrogen will not leak out during the release of hydrogen. The purpose of setting the fixed disc 5 is to make it more stable for the experimenter to take and move when using it.

[0040] The connecting sleeve 2 is annular and cylindrical, and adhesive is provided at the connection between the connecting sleeve 2 and the dish body 4.

[0041] The presence of the filter membrane 3 can isolate the cells from contact with hydrogenated coral calcium to avoid affecting cell growth, and can also allow water molecules to pass through the filter membrane into the placement cavity a9, so that the cells grow normally on the filter membrane 3.

[0042] The inner diameter of the dish cover 1 is larger than the outer diameter of the connecting sleeve 2 , and the outer diameter of the dish cover 1 is not larger than the outer diameter of the fixed disc 5 .

[0043] like Figure 5As shown, a support sheet 7 is fixedly connected to the inner wall of the top cover of the dish cover 1. The position of the support sheet 7 corresponds to the upper edge of the connecting sleeve 2, that is, the support sheet 7 is located directly above the upper edge of the connecting sleeve 2, and the distance from the support sheet 7 to the upper edge of the connecting sleeve 2 is smaller than the distance from the lower edge of the dish cover 1 to the fixed disc 5. When the dish cover 1 and the connecting sleeve 2 are buckled, the support sheet 7 can be added between the top of the inner wall of the dish cover 1 and the upper edge of the connecting sleeve 2 device to prevent the dish cover 1 from falling, so that there is no airtight contact between the dish cover 1 and the fixed disc 5, and there is a certain air circulation gap, so that the necessary gas when culturing cells passes through the gap and provides the necessary gas environment for cell growth.

[0044] A method for preparing a hyperoxic culture medium is as follows: a sterile culture medium is poured into a plastic sealed bottle, and the bottle and an 8F-5AW medical molecular sieve oxygen generator are placed in a sterile operating room for oxygenation to obtain a hyperoxic culture medium.

[0045] The present invention uses the above-mentioned cell culture device to provide a method for enriching the cell culture medium with hydrogen, which is specifically implemented according to the following steps:

[0046] The liquid culture medium or the high oxygen culture medium is placed in the placement cavity b10 of the connecting sleeve 2, and the hydrogenated coral calcium is added into the placement cavity a9. The water molecules in the liquid culture medium or the high oxygen culture medium contact with the hydrogenated coral calcium through the filter membrane 3 to generate hydrogen and merge into the liquid culture medium or the high oxygen culture medium.

[0047] The top support sheet 7 of the dish cover 1 ensures that the circular dish cover 1 and the cylindrical connecting sleeve 2 are not completely sealed during cell culture, ensuring that the placement cavity b10 can exchange gas with the gas in the cell culture box to provide the necessary gas environment for cell growth.

[0048] The working principle of the present invention is: by utilizing the characteristic that hydrogenated coral calcium stably and persistently releases hydrogen when in contact with water, the filter membrane 3 is used to filter the hydrogenated coral calcium added to the placement chamber a9, and the placement chamber a9 for placing the hydrogenated coral calcium is isolated from the placement chamber b10 for culturing cells to prevent the hydrogenated coral calcium from entering the placement chamber b10. When conducting cell experiments, a common liquid culture medium such as DMEM or the high oxygen culture medium prepared above is added to the placement chamber b10, and water molecules will contact the hydrogenated coral calcium through the filter membrane 3 to generate hydrogen. The hydrogen is continuously released and integrated into the culture medium, so that the hydrogen molecules in the original culture medium are dissolved to form a hydrogen-rich culture medium.

[0049] The use process of the present invention is as follows: before use, the user adds a certain dose of hydrogenated coral calcium into the placement cavity a9, covers the placement cavity a9 with a filter membrane 3, and threadedly connects the lower thread 8 on the inner wall surface of the dish body 4 with the upper thread 6 on the lower outer wall surface of the connecting sleeve 2, seals the circular filter membrane 3, the annular cylindrical connecting sleeve 2, and the circular dish body 4, and then adds a certain amount of cell culture medium into the placement cavity b10 to culture cells, and finally covers the circular dish cover 1.

[0050] The effect of hydrogen enrichment of cell culture medium using the above cell culture device was verified:

[0051] Different concentrations of hydrogenated coral calcium, 5g / L, 7.5g / L and 10g / L, were used to culture the medium. The hydrogen content of the medium was measured at intervals of 1 hour. The hydrogenated coral calcium with concentrations of 5g / L, 7.5g / L and 10g / L was mixed with distilled water at room temperature, and then the hydrogen content in the water was measured. During the experiment, we selected the same distilled water, the same temperature, the same measuring instrument, and the same person to measure the same technical standards to ensure that only the content of hydrogenated coral calcium was the variable.

[0052] like Figure 6 As shown in the figure, it is a line graph of the hydrogen content released by hydrogenated coral calcium in water at different concentrations of 5g / L, 7.5g / L and 10g / L. We mixed hydrogenated coral calcium with distilled water at room temperature at 5g / L, 7.5g / L and 10g / L, and then measured the hydrogen content in the water. During the experiment, we selected the same distilled water, the same temperature, the same measuring instrument, and the same person to perform the same technical standard measurement to ensure that only the content of hydrogenated coral calcium was a variable. The resulting data curve is shown in the figure below. Figure 6 As shown, from Figure 6 It can be seen intuitively that with the passage of time, all three curves show an upward trend from 0h and reach a peak at 6h. In the following period of time, the hydrogen content of 7.5g / L and 10g / L shows a downward trend, and the upward trend of the hydrogen content graph of 5g / L slows down, while the hydrogen content is still at a very high value. According to the curves of hydrogenated coral calcium of different concentrations, it can be seen that the hydrogen content of 7.5g / L and 10g / L is significantly higher than that of 5g / L. Comparing the graphs of 7.5g / L and 10g / L, it can be seen that the hydrogen content curves of these two concentrations have similar trends and the hydrogen content is close.

[0053] According to the graphs of 5g / L, 7.5g / L and 10g / L, it can be seen that within a certain range of concentrations, the hydrogen content will increase with the increase of the concentration of hydrogenated coral calcium. However, as the concentration of hydrogenated coral calcium increases further, as can be seen from the graphs of 7.5g / L and 10g / L, the hydrogen content does not completely show a positive correlation with the concentration of hydrogenated coral calcium. And according to the experimental data curve, the data graph of 7.5g / L is very representative. Therefore, 7.5g / L was also selected as a representative in subsequent measurements.

[0054] From the above experiments, we can conclude that hydrogenated coral calcium can continuously and stably release hydrogen when in contact with liquid water, and there is a certain degree of saturation in the process of hydrogenated coral calcium producing hydrogen when in contact with liquid water. That is, when the content of hydrogenated coral calcium in water reaches a certain concentration, the concentration of hydrogenated coral calcium increases and the hydrogen content does not always show a positive correlation increase.

[0055] When getting Figure 6 After the experimental results shown, we took hydrogenated coral calcium with a concentration of 7.5g / L and combined it with liquid culture medium to produce hydrogen. According to the method of measuring once every 1h, during the experiment, we selected a brand new liquid culture medium and placed it in a cell CO2 box for culture, that is, 37℃, 5% CO2 for measurement. The same measuring instrument and the same person performed the same technical standard measurement to ensure that there was only one variable, the content of hydrogenated coral calcium. The results obtained are as follows Figure 7 As shown in the figure, according to the experimental data, the hydrogen content was 383ppb at 0h, and then the hydrogen content showed an upward trend, and showed a peak of 535ppb at 6h and 7h after the start of measurement, and then the hydrogen content decreased over time and reached 425ppb after 24h. This experiment confirmed that hydrogenated coral calcium can not only combine with water to stably release hydrogen, but also, compared with distilled water, the culture medium and hydrogenated coral calcium can stably produce hydrogen for a long time and at a higher content.

[0056] During the experiment, we also selected hydrogen-rich water liquid at room temperature, used the same measuring instruments, and the same person to measure the hydrogen content using the same technical standards. The results are as follows: Figure 8 As shown in the figure, the hydrogen content of hydrogen-rich water at 0h is 700ppb. We placed the hydrogen-rich water in a beaker and measured the hydrogen every 10 minutes. During the measurement, we measured at room temperature. The same measuring instrument and the same person performed the same technical standard measurement, and the results were as follows Figure 8 According to Figure 8From the experimental data trend chart, it can be seen that the hydrogen content of hydrogen-rich water has been decreasing over time, and it has completely dropped to 0ppb at 120 minutes. This experiment proves that hydrogen-rich water prepared by the hydrogen filling method can only keep hydrogen in the liquid for a very short time, and cannot guarantee the hydrogen content in the liquid for a long time. This is also the disadvantage of the traditional hydrogen filling method to prepare hydrogen-rich liquid, and it is also the reason why we gave up the hydrogen filling method to prepare hydrogen-rich culture medium.

[0057] Based on the above experiments, Fig. 9 For the above Figure 6 , Figure 7 and Figure 8 The experimental results of the reaction between 7.5g / L hydrogenated coral calcium and water are compared. Fig. 9 It can be seen that the hydrogen content of hydrogen-rich water has reached 0ppb in 2 hours, and compared with 7.5g / L hydrogenated coral calcium, the liquid combined with hydrogenated coral calcium can maintain a higher content of hydrogen for a long time, and compared with water, the hydrogen content produced by the combination of hydrogenated coral calcium and liquid culture medium can be higher. So we can conclude that the present invention combines hydrogenated coral calcium with liquid culture medium, which can stably enrich the culture medium with hydrogen for a long time. This hydrogen enrichment method is superior to the traditional hydrogen enrichment method.

[0058] The effect of the high oxygen and hydrogen-rich culture medium prepared in this application was verified:

[0059] The cell culture device of the present invention is used to oxygenate the liquid culture medium and physiological saline at room temperature, respectively, and the dissolved oxygen content is measured together with the high-oxygen water and ordinary tap water purchased from the market. During the experiment, we selected the same ambient temperature, the same liquid volume, the same measuring instrument, and the same person to perform the same technical standard measurement to ensure that there is only one variable, that is, different oxygen-containing liquids.

[0060] The results of the test are as follows Fig.10As shown in the figure, it can be seen from the data in the figure that the three curves of oxygen-enriched culture medium, oxygen-enriched saline, and commercially purchased high-oxygen water (purchased from Zhejiang Oxygen Dian Beverage Co., Ltd.) all show a downward trend over time. The dissolved oxygen content at 0 min is 25.82 mg / L, 21.96 mg / L, and 18.01 mg / L, respectively. At 180 min, the three curve values ​​are 8.55 mg / L, 9.38 mg / L, and 8.45 mg / L, respectively, while the dissolved oxygen content of tap water has always been at a value of 8.49 mg / L. Comparing the data curves of oxygen-enriched culture medium and oxygen-enriched saline, it can be seen that the dissolved oxygen content of oxygen-enriched culture medium is significantly greater than that of oxygen-enriched saline at 0 min, and then both curves show a downward trend, and after 180 min, the curve data gradually tends to stabilize. Comparing the data curves of oxygen-enriched saline and hyperoxia water, it can be seen that the trends of the two curves are similar. At 0 min, the dissolved oxygen content of oxygen-enriched saline is significantly higher than that of hyperoxia water. As time goes by, the curves show a downward trend, and at 180 min, the curve data gradually becomes stable.

[0061] From the above experimental data, we can conclude that the dissolved oxygen content in the oxygen-enriched culture medium and oxygen-enriched saline prepared by oxygenation is significantly higher than that in the high-oxygen water purchased on the market, and the dissolved oxygen content of the three oxygen-containing liquids shows a decreasing trend over time, and at 180 minutes the oxygen content change trends of the three gradually stabilized and the oxygen content was still above 8 mg / L, which proves the feasibility of our invention.

[0062] In order to further confirm the advantages and feasibility of this high-oxygen hydrogen-rich culture medium, we oxygenated the liquid culture medium at room temperature. In order to better combine the experimental methods of the experimenter, we placed the oxygenated liquid culture medium and the unoxygenated ordinary culture medium in culture bottles and culture dishes and placed them in a 37°C, 5% CO2 cell culture incubator to measure dissolved oxygen. During the experiment, we selected the same ambient temperature, the same liquid volume, the same measuring instrument, and the same person to perform the same technical standard measurement.

[0063] The experimental results are as follows Fig.11As shown in the figure, it can be seen that by comparing the dissolved oxygen content curve of the oxygen-enriched medium in the culture bottle and the dissolved oxygen content curve of the oxygen-enriched medium in the culture dish, the dissolved oxygen content is 25.82 mg / L at 0 min. As time goes by, both curves show a downward trend and the curve trends are almost the same. At 180 min, the trend tends to be stable, and the dissolved oxygen content is 5.64 mg / L and 5.53 mg / L respectively. The dissolved oxygen content curves of the ordinary culture medium in the culture bottle and the ordinary culture medium in the culture dish show that the trends of the two curves are almost the same. At 0 min, the dissolved oxygen content is 8.20 mg / L, and the curve shows a relatively slow downward trend. At 40 min, the curve trend gradually tends to be stable. At 180 min, the dissolved oxygen content is 5.31 mg / L and 5.27 mg / L respectively.

[0064] From the above experimental data, we can conclude that the dissolved oxygen content of oxygen-enriched culture medium can maintain a higher content for a longer time in the cell culture incubator. Compared with ordinary liquid culture medium, oxygen-enriched culture medium can contain high content of dissolved oxygen in different environments. And the oxygen content is similar in different cell culture devices.

[0065] Combination Fig.10 and Fig.11 It can be seen that the oxygen-enriched culture medium obtained by the present invention can maintain a certain dissolved oxygen content for a long time no matter in a room temperature environment or in a special environment of a cell culture box.

Claims

1. A cell culture device, characterized in that: The invention comprises a dish cover (1) and a dish body (4) arranged in a vertical direction, a connecting sleeve (2) being arranged between the dish cover (1) and the dish body (4), the dish cover (1) and the dish body (4) being both cylindrical, the connecting sleeve (2) being annular, a fixing disc (5) being arranged on the outer wall of the connecting sleeve (2), a supporting sheet (7) being fixedly connected to the inner wall of the top cover of the dish cover (1), the supporting sheet (7) being located just above the upper edge of the connecting sleeve (2), the supporting sheet (7) being connected to the upper edge of the connecting sleeve (2) The distance between the edges is smaller than the distance between the lower edge of the dish cover (1) and the fixed disc (5); a placement cavity a (9) is formed inside the dish body (4); a filter membrane (3) is arranged inside the placement cavity a (9); the filter membrane (3) is circular; the diameter of the filter membrane (3) is larger than the inner diameter of the placement cavity a (9); a placement cavity b (10) is formed inside the connecting sleeve (2); the dish cover (1), the connecting sleeve (2), the dish body (4) and the fixed disc (5) are all made of transparent polystyrene material.

2. A cell culture device according to claim 1, characterized in that: The inner diameter of the dish cover (1) is larger than the outer diameter of the connecting sleeve (2), and the outer diameter of the dish cover (1) is not larger than the outer diameter of the fixed disc (5).

3. A cell culture device according to claim 1, characterized in that: An adhesive is provided at the connection between the connecting sleeve (2) and the dish body (4).

4. A cell culture device according to claim 1, characterized in that: An upper thread (6) is provided on the outer side of the bottom of the connecting sleeve (2), and the upper thread (6) is located at the bottom of the fixed disc (5). A lower thread (8) is provided on the inner side wall of the dish body (4), and the upper thread (6) and the lower thread (8) match each other.

5. A method for enriching a cell culture medium with hydrogen, characterized in that: Using the device described in claim 1, the method is implemented specifically according to the following steps: The liquid culture medium is placed in the placement chamber b (10) of the connecting sleeve (2), and hydrogenated coral calcium is added into the placement chamber a (9). The water molecules in the liquid culture medium pass through the filter membrane (3) to contact the hydrogenated coral calcium to generate hydrogen gas which is then incorporated into the liquid culture medium to obtain a hydrogen-rich culture medium.

6. A method for enriching a cell culture medium with hydrogen according to claim 5, characterized in that: The liquid culture medium can also be replaced by a hyperoxic culture medium, which is prepared according to the following method: a sterile culture medium is poured into a plastic sealed bottle, and the bottle is placed in a sterile operating room together with a medical oxygen generator for oxygenation to obtain a hyperoxic culture medium.

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