A kind of based on double carbon source synchronization supply ginseng plantlet stable isotope 13 C labeling method

Abstract

This invention provides a stable isotope method for tissue culture plants of Codonopsis pilosula based on simultaneous supply of dual carbon sources. 13 C-labeling method. This is achieved by synchronously introducing [the method] under tissue culture conditions. 13 High abundance was obtained by using C-labeled soluble and gaseous carbon sources. 13 C-labeled Codonopsis pilosula plants and their polysaccharides, to solve the problem of not being able to obtain polysaccharides from traditional Chinese medicine using existing technologies. 13 The problems of uncontrollable C labeling sources, unstable labeling efficiency, and difficulty in obtaining structurally consistent labeled samples provide a reliable material basis for subsequent metabolic tracing and related research.

Description

Technical Field

[0001] This invention relates to the fields of plant physiology and biochemistry, and in particular to a stable isotope for tissue culture plants of Codonopsis pilosula. 13 A C-labeling method and its dedicated apparatus, which can be used to obtain high abundance 13 C-labeled *Pseudostellaria heterophylla* plants and *Pseudostellaria heterophylla* polysaccharides. This method and apparatus can be used to study the biosynthesis process and structural characteristics of *Pseudostellaria heterophylla* polysaccharide PF40. Background Technology

[0002] Traditional Chinese medicine (TCM), a precious cultural heritage of the Chinese nation, boasts a long history and rich clinical application experience. The active ingredients in TCM, especially polysaccharides, have been proven to have significant pharmacological effects and clinical efficacy. However, the absorption, distribution, metabolism, and excretion processes of these active ingredients in the body are not yet fully understood, which to some extent limits the in-depth research into the mechanisms of action of TCM and the further development of its clinical application.

[0003] To delve deeper into the in vivo processes of active components in traditional Chinese medicine (TCM), isotope labeling technology has become an important research tool. Through isotope labeling, combined with techniques such as isotope-resolved metabolomics and DNA-SIP (stable isotope probing), the metabolic pathways and transformation processes of active TCM components in vivo can be traced. However, currently used radionuclide labeling methods have many limitations, such as poor stability, radiation hazards, high requirements for equipment and facilities, and high costs.

[0004] To address the shortcomings of existing technologies, this invention provides a simple, safe, and stable isotope labeling method. During the cultivation of *Pseudostellaria heterophylla* tissue culture seedlings, by providing... 13 C-glucose and 13 Using C-CO2 as a carbon source, efficient processing of Codonopsis pilosula plants was achieved. 13 C-marking. This method not only improves 13 This method not only improves the labeling rate of C but also avoids the potential risks associated with radionuclides, providing a new technical means for the metabolic research of active ingredients in traditional Chinese medicine. Summary of the Invention

[0005] The purpose of this invention is to provide a stable and controllable stable isotope profile of Codonopsis pilosula tissue culture plants. 13 The C-labeling method, through synchronous introduction under tissue culture conditions... 13 High abundance was obtained by using C-labeled soluble and gaseous carbon sources. 13 C-labeled Codonopsis pilosula plants and their polysaccharides, to solve the problem of not being able to obtain polysaccharides from traditional Chinese medicine using existing technologies. 13 The problems of uncontrollable C labeling sources, unstable labeling efficiency, and difficulty in obtaining structurally consistent labeled samples provide a reliable material basis for subsequent metabolic tracing and related research.

[0006] Existing studies on the isotope labeling of polysaccharides from traditional Chinese medicines mostly rely on exogenous chemical synthesis or in vitro labeling methods, which cannot truly reflect their natural biosynthesis process in plants; at the same time, traditional field or pot cultivation conditions are not suitable for this purpose. 13 C-labeling suffers from numerous environmental variables, uncontrollable carbon sources, and poor reproducibility, making it difficult to obtain stable and uniformly labeled polysaccharide products. Furthermore, conventional plant tissue culture systems typically use sucrose as the primary carbon source, and commercially available alternatives are lacking. 13 The use of carbonyl sucrose for labeling presents practical difficulties in achieving stable isotope labeling under tissue culture conditions. Therefore, there is an urgent need for a method that utilizes readily available carbonyl sucrose within a controlled tissue culture system. 13 A carbon source and a method for achieving stable and reproducible labeling are needed to meet the needs of research on polysaccharides in traditional Chinese medicine.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: Stable isotopes of a tissue culture plant of Codonopsis pilosula 13 The C-marking method includes the following steps: (1) A tissue culture system for Codonopsis pilosula was established under aseptic conditions. Aseptic seedling explants of Codonopsis pilosula were inoculated into a solid culture medium containing glucose and cultured. The glucose was the soluble carbon source of the culture medium. (2) During the culture process, a sterile carbon dioxide replenishment system is used to replenish carbon dioxide into the gas phase space of the tissue culture container; (3) During the same culture cycle, the glucose in the culture medium is replaced with 13 C-labeled glucose, while replacing the supplemented carbon dioxide with 13 C-labeled carbon dioxide was cultured using a dual-carbon-source simultaneous supply method to obtain... 13 C-labeled micro-tuberous roots of Codonopsis pilosula; (4) From the above 13 C-labeled extract from Codonopsis pilosula micro-tuberous roots 13 C-labeled Codonopsis pilosula polysaccharide PF40.

[0008] Furthermore, the culture medium does not contain sucrose, and the concentration of glucose is 5-45 g / L.

[0009] Furthermore, the aforementioned 13 C-labeled glucose is 13 C6-glucose.

[0010] Furthermore, the aforementioned 13 C-labeled carbon dioxide through 13 C-NaHCO3 reacts with sulfuric acid to produce the product, which is then introduced into the tissue culture container.

[0011] Furthermore, the effective gas phase volume of the tissue culture container is 200 mL, and a single replenishment increases the carbon dioxide concentration in the tissue culture container from 400 ppm to 1500 ppm.

[0012] Furthermore, the carbon dioxide replenishment frequency is once every 1-7 days.

[0013] Furthermore, the sterile carbon dioxide replenishment system is equipped with 0.22 μm hydrophobic filter membrane units at the gas generation outlet, the branch flow path, and the end before entering each tissue culture container.

[0014] Furthermore, each tissue culture container is equipped with a breathable needle on its cap, which is connected to a 0.22 μm hydrophobic filter membrane unit to achieve pressure balance inside and outside the tissue culture container and maintain a sterile barrier.

[0015] Furthermore, the aseptic nature of the method 13 A CO2 supplementation system includes: (a) a gas generating module for generating carbon dioxide through the reaction of acid and bicarbonate; (b) a gas filtration and delivery module, the outlet of which is connected to the gas filtration and delivery module, the gas filtration and delivery module including at least one 0.22 μm hydrophobic filter membrane unit and sterile tubing; (c) a manifold distribution module connected to the gas filtration and delivery module, the manifold distribution module having multiple branch lines and each branch line having an independent valve; and (d) an end-connection module connected to each branch line for introducing carbon dioxide filtered through the 0.22 μm hydrophobic filter membrane into multiple tissue culture containers.

[0016] Furthermore, the gas generating module includes a container for holding sulfuric acid and a syringe interface for adding bicarbonate solution.

[0017] Furthermore, each branch pipe of the manifold diversion module is equipped with a 0.22 μm hydrophobic filter membrane unit.

[0018] Furthermore, each tissue culture container has a central opening in its cap and a vent connector installed to connect to the end access module, allowing carbon dioxide to enter the gas phase space of the tissue culture container through the vent connector.

[0019] Furthermore, each tissue culture container is equipped with a breathable needle that is connected to a 0.22 μm hydrophobic filter membrane unit, serving as an exhaust valve for the tissue culture container to achieve pressure balance.

[0020] Furthermore, the sterile tubing is a medical sterile infusion tubing, and each connector is made of a material that can be sterilized at high temperatures.

[0021] Furthermore, the gas filtration and delivery module further includes a quick-connect valve for quickly connecting and disconnecting the gas pipeline without compromising sterility.

[0022] The advantages of this invention are: 1. This invention introduces the simultaneous [introduction] under tissue culture conditions. 13 The use of C-labeled soluble and gaseous carbon sources allows *Codonopsis pilosula* to obtain stable isotopes within the same culture cycle. 13 The C-labeled micro-roots and their polysaccharide products have a clear technical route and a controllable operation process.

[0023] 2. This invention uses glucose as the carbon source for the culture medium, thus avoiding the lack of commercially available sucrose. 13 The implementation difficulties introduced by C-labeling reagents are addressed by selecting carbon isotopes with clearly defined sources, which are suitable for stable isotope labeling operations under routine experimental conditions.

[0024] 3. This invention uses a tissue culture system for labeling, which provides a stable culture environment with few external interference factors, making it easier to obtain polysaccharide samples of Codonopsis pilosula with uniform isotope abundance and good reproducibility.

[0025] 4. Experimental verification shows that the method obtained using this invention... 13 C-labeled Codonopsis pilosula polysaccharides maintain consistency with unlabeled polysaccharides in terms of major functional group composition and polysaccharide backbone structure, thus preserving their original biological activity.

[0026] 5. The present invention has a wide range of applications, not only applicable to tissue culture seedlings of Codonopsis pilosula. 13 C labeling can also be extended to isotope labeling research on other medicinal plants. Attached Figure Description

[0027] Figure 1 This is a physical image of the carbon dioxide replenishment device in the tissue culture bottle provided in Embodiment 1 of the present invention.

[0028] Figure 2 This is a schematic diagram of the structure of the carbon dioxide replenishment device in the tissue culture bottle provided in Embodiment 1 of the present invention.

[0029] Figure 3 The method for obtaining the tuberous roots and stems / leaves of *Codonopsis pilosula* using the method of the present invention is provided in Embodiment 4 of the present invention. 13 Comparison of C values.

[0030] Figure 4 Fourier transform infrared spectra of tissue-cultured Codonopsis pilosula polysaccharide (A) and natural Codonopsis pilosula polysaccharide (B) provided in Example 5 of this invention. Detailed Implementation

[0031] To illustrate the implementation of this invention, several embodiments are provided below to describe the tissue culture seedlings of *Pseudostellaria heterophylla*. 13 Applications of C-labeling methods and apparatus.

[0032] Example 1: CO2 replenishment device in tissue culture flasks Gas Generation and Introduction System: The glass bottle on the right side of the device serves as the gas generation module 1, containing 1 M H2SO4 solution 2. A three-way connector is connected to the top (red pressure-resistant cap). A syringe 3 is connected to one side to drip sodium bicarbonate solution as needed, causing a chemical reaction with the reactants in the bottle to generate carbon dioxide. The generated CO2 first enters the conduit 4 through the top connector. The conduit is equipped with a green gas filter unit 5 (hydrophobic membrane unit) and a quick-connect valve to ensure preliminary purification and buffering of the gas before introduction. Downstream, CO2 is delivered to multiple tissue culture bottles 7 on the left side via a transparent sterile tubing 6. To ensure uniform gas supply among the bottles, a blue and green manifold distribution module 8 is designed. This module has multiple branch lines, each with an independent valve (a knob valve 9) for flexible control of the ventilation and flow rate of individual bottles. The tissue culture flask has a centrally located opening in its cap, a vent connector installed, and a gas line 10 connected to it. CO2 is evenly distributed into the flask's interior, promoting photosynthesis in the plants. The entire introduction process achieves a complete pathway from gas production and diversion to entry into the culture flask. Figure 1 and Figure 2 As shown.

[0033] Sterilization and Aseptic Protection: The key to the entire system is maintaining the sterility of the culture system. First, green 0.22 μm hydrophobic filter membrane units are installed at the gas generator bottle outlet and at each branch line. These membranes effectively block bacterial and fungal spores while allowing gas to pass freely. Transparent tubing uses sterile medical infusion tubing, and all connectors are made of heat-sterilizable materials, ensuring readily available experimental materials. The diversion valves are designed externally to reduce the risk of cross-contamination during operation. Furthermore, the gas entering each culture bottle only connects to the internal space after passing through the final 0.22 μm filter membrane, ensuring that even if microorganisms or acid fumes remain upstream, they will not enter the culture system. In this device, a medical needle is inserted into the top of the cap of each tissue culture bottle and connected to the 0.22 μm hydrophobic filter membrane unit. The core purpose of this design is to address the dynamic changes in gas pressure inside the bottle: as CO2 is continuously introduced through the tubing, the volume and pressure of the gas inside the bottle gradually increase. Without a vent, this could easily lead to the bottle cap loosening, tubing leaks, or even the culture medium being ejected. The needle vent acts as a single-bottle exhaust valve, utilizing the microporous structure of the hydrophobic filter membrane to allow free gas exchange while maintaining a sterile barrier, thus ensuring a balance between the pressure inside and outside the bottle. Because the filter membrane is hydrophobic, even if there is condensation or culture medium vapor inside the bottle, it will not permeate through the membrane into the outside, greatly reducing the risk of liquid contamination and aerosol escape. Through the combination of "multi-stage filtration + independent valves + sterile tubing," this device can effectively maintain a long-term sterile culture environment while ensuring efficient carbon dioxide replenishment.

[0034] Example 2: Optimization of glucose addition in culture medium Using uniformly grown aseptic seedlings of *Pseudostellaria heterophylla* as material, stem segments of consistent length containing a single internode were selected as explants. MS solid medium was used as the base, without added sucrose, with 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, and 45 g / L glucose added as the sole soluble carbon source. The pH of the medium was adjusted to 5.8, and the medium was autoclaved at 121 ℃ for 20 min before use. Five stem segments were inoculated into each bottle, with 10 bottles per treatment as biological replicates. The culture conditions were set as follows: temperature 25 ℃, light intensity 2000 lx, and photoperiod 16 h / 8 h. The moisture content of the medium in each bottle was kept consistent during culture, and no additional nutrient solution was added. After 45 days of culture, samples were taken uniformly to measure plant height, tuber diameter, and tuber fresh weight. Plant height was measured as the length from the base to the stem tip, tuber diameter was measured using calipers to determine the maximum transverse diameter, and tuber weight was measured using an analytical balance to determine the fresh weight of individual tubers. All data are expressed as mean ± standard deviation and are used for subsequent statistical analysis.

[0035] Table 1 shows that different glucose concentrations significantly affected the vegetative growth and micro-tuber formation of tissue-cultured *Pseudostellaria heterophylla*. With increasing glucose concentration in the culture medium, plant height generally decreased, reaching its highest point (8.52 cm) at 5 g / L. However, plant height decreased significantly in the 30-45 g / L range, indicating that higher carbon source levels inhibited the elongation of the aboveground parts. Conversely, tuber diameter and weight increased significantly with increasing glucose concentration, reaching stable high values ​​at concentrations of 30 g / L and above. The 5 g / L treatment group had the lowest tuber diameter and weight, while the 30 g / L treatment group had significantly higher tuber diameter and weight than the low-concentration group. When the glucose concentration was further increased to 40-45 g / L, tuber diameter and weight no longer increased significantly, indicating that tuber enlargement tended to saturate in this range. Statistical analysis showed that different letters represented significant differences (P<0.05). Considering the combined effects of plant height inhibition and tuber enlargement, 30 g / L glucose performed better in promoting micro-tuber formation and maintaining the overall plant growth balance.

[0036] Table 1. Effects of different glucose concentrations on plant height, tuber diameter, and tuber weight of tissue-cultured Codonopsis pilosula.

[0037] Example 3: Optimization of CO2 replenishment frequency Based on the determination that the optimal glucose concentration was 30 g / L, the effect of CO2 supplementation frequency on micro-tuber formation was further investigated. CO2 was generated using a sodium bicarbonate-sulfuric acid reaction system in a closed container, and the generated CO2 was directly introduced into the tissue culture flasks via an acid-resistant conduit. The effective gas volume of each flask was 200 mL, and the target for a single supplementation was to increase the CO2 concentration in the flask from the background level to approximately 1500 ppm. Supplementation frequencies were set as no supplementation, supplementation once every 1 day, supplementation once every 3 days, and supplementation once every 7 days, with all other culture conditions remaining consistent across treatments. Each treatment was replicated in 10 flasks, with 5 stem segments inoculated into each flask. Culture conditions were 25 ℃, light intensity 2000 lx, and photoperiod 16 h / 8 h. After 45 days of culture, samples were taken uniformly to measure plant height, tuber diameter, and tuber weight, using the same methods as in the glucose concentration experiment. The effect of CO2 supply rhythm on microtube formation was assessed by comparing changes in plant growth and microtube enlargement indices under different CO2 supplementation frequencies.

[0038] The results showed that different CO2 supplementation frequencies significantly affected the growth and micro-tuber formation of tissue-cultured *Pseudostellaria heterophylla*. Under conditions of no CO2 supplementation or low supplementation frequency, plant height did not differ significantly among treatments, with an overall fluctuation range of 7.26–9.04 cm, indicating that CO2 supplementation had a limited effect on aboveground elongation. In contrast, tuber diameter and weight showed a more sensitive response to CO2 supplementation frequency. Specifically, the tuber diameter and weight in the higher CO2 supplementation frequency treatment group were significantly higher than the control group, with the highest tuber diameter reaching 0.31 cm and tuber weight reaching 0.112 g, significantly higher than the low-frequency or no-supplementation treatment (P<0.05). When the supplementation frequency was reduced to 7 days / time, both tuber diameter and weight showed a decreasing trend, and the difference between the two treatments was no longer significant, indicating that insufficient CO2 supply limited the continuous input of carbon assimilation to underground storage organs. In summary, appropriately increasing the CO2 supplementation frequency is beneficial for promoting micro-tuber enlargement and biomass accumulation, but its promoting effect is significantly weakened when the supplementation interval is too long.

[0039] Table 2. Effects of different CO2 supplementation frequencies on plant height, tuber diameter, and tuber weight of tissue-cultured Codonopsis pilosula.

[0040] Example 4: Verification of the effect of two-carbon labeling Experimental design: for comparison using alone 13 C-glucose, used alone 13 The combined use of C-CO2 and dual carbon sources for the effects of Codonopsis pilosula 13To investigate the effect of C labeling efficiency, sterile stem segments of *Codonopsis pilosula* seedlings with uniform growth (containing a single internode and of uniform length) were selected as explants and inoculated into MS solid medium for a parallel control experiment. The medium used glucose as the sole soluble carbon source, with a fixed concentration of 30 g / L, and three treatment conditions were set: (1) only glucose was used as the sole soluble carbon source. 13 C-glucose group: Culture medium used 13 C6-glucose (atomic abundance ≥ 99%) 30 g / L; (2) only 13 C-CO2 group: Culture medium used 12 C-glucose 30 g / L, CO2 supply from 13 C-NaHCO3 (atomic abundance ≥ 99%) reacts with 1 M H2SO4 to produce 13 CO2; (3) Dual carbon source group: culture medium used 13 C6-glucose 30 g / L, while CO2 supply is from 13 C-NaHCO3 reacts with 1 M H2SO4 to produce 13 CO2 supplementation was performed using the device designed in this invention. The effective gas volume of the tissue culture bottle was 200 mL. A single supplementation increased the CO2 concentration in the bottle from approximately 400 ppm to 1500 ppm. The supplementation frequency was fixed at once every 3 days. Before each supplementation, the airtightness of the tubing was checked, and aeration was performed in a clean bench to avoid contamination. Five stem segments were inoculated into each bottle, and 10 biological replicates were set up for each treatment. The culture conditions were 25 ℃, light intensity of 2000 lx, and photoperiod of 16 h / 8 h. After 45 days of culture, the tuberous root and stem / leaf tissues were collected, flash-frozen in liquid nitrogen, freeze-dried, and ground through a 100-mesh sieve. 1-3 mg of the sample was weighed, packed tightly, and sealed in a tin cup. The sample was then analyzed by elemental analysis-isotope ratio mass spectrometry (EA-IRMS). 13 C / 12 C ratio and converted to δ 13 The C-value (based on VPDB) is used to evaluate different 13 The marking efficiency and tissue distribution differences of C carbon source supply strategies in tubers and stems / leafs.

[0041] exist 13 C-glucose and 13 Under combined CO2 supplementation conditions, the tuberous roots and stems / leaves of *Pseudostellaria heterophylla* 13 C abundance was at the highest level in both tubers and leaves (43201 ± 3241‰, 46201 ± 4215‰), significantly higher than that in stems and leaves alone. 13 C-glucose treatment (roots 38220 ± 2909‰; stems and leaves 40502 ± 3640‰) and only 13CO2 treatment (roots 12604 ± 1904‰; stems and leaves 18904 ± 2707‰). These results indicate that simultaneous dual-carbon source labeling can simultaneously cover both "exogenous soluble carbon source input" and "photosynthetic carbon fixation input" pathways within the same culture cycle, thereby improving the overall tissue quality. 13 The enrichment of C results in a combined improvement in labeling efficiency and labeling strength.

[0042] Table 3 Differences 13 C-carbon source feeding strategy for the tuberous roots and stems of Codonopsis pilosula 13 Influence of C abundance

[0043] Example 5: Physicochemical Properties Analysis of Codonopsis pilosula Polysaccharide 13C-PF40 To verify the effectiveness of the Codonopsis pilosula polysaccharide prepared by this method 13 Whether the physicochemical properties of C-PF40 change, this study uses conventional Codonopsis pilosula polysaccharide as an example. 12 Using C-PF40 as a control, the functional group characteristics of the two were compared and analyzed using Fourier transform infrared spectroscopy (FT-IR). 13 C-PF40 and 12 C-PF40 samples were dried in a vacuum drying oven at 60 °C for 12 h to remove adsorbed water. Then, 2 mg of each sample was weighed and thoroughly ground with 200 mg of dried KBr (spectrally pure) (mass ratio 1:100). The mixture was then pressed into transparent sheets at 10 MPa for 1 min. Spectra were acquired at room temperature using an FT-IR spectrometer (Bruker Tensor II, Germany), with a scanning range of 4000–400 cm⁻¹. -1 4 cm resolution -1 The scan was performed 32 times, with blank KBr pellets used as background subtraction. Each sample was prepared independently and analyzed three times. FT-IR results show that 13 C-PF40 and regular 12 The infrared spectral characteristics of C-PF40 are highly consistent. Both are at approximately 3400 cm⁻¹. -1 A broad and strong absorption peak for the OH stretching vibration appears at approximately 2920 cm⁻¹. -1 The characteristic peak of CH stretching vibration appears at 1600-1650 cm⁻¹. -1 It exhibits an absorption signal associated with bound water within the range; simultaneously, in the 1200-800 cm⁻¹ range... -1 The fingerprint region consistently exhibits typical COC and CO vibrational absorption peaks characteristic of polysaccharides, with consistent peak positions and relative intensities. These results demonstrate that the fingerprint obtained using the method of this invention... 13The main functional group composition and polysaccharide backbone structure of C-PF40 remained unchanged.

[0044] The above embodiments are merely examples, and adjustments can be made according to specific needs in actual applications. The apparatus and method of the present invention have broad applicability and can be used for isotope labeling studies of other medicinal plants.

[0045] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. Stable isotopes of a tissue-cultured strain of *Codonopsis pilosula* 13 The C-marking method is characterized by... Includes the following steps: (1) A tissue culture system for Codonopsis pilosula was established under aseptic conditions. Aseptic seedling explants of Codonopsis pilosula were inoculated into a solid culture medium containing glucose and cultured. The glucose was the soluble carbon source of the culture medium. (2) During the culture process, a sterile carbon dioxide replenishment system is used to replenish carbon dioxide into the gas phase space of the tissue culture container; (3) During the same culture cycle, the glucose in the culture medium is replaced with 13 C-labeled glucose, while replacing the supplemented carbon dioxide with 13 C-labeled carbon dioxide was cultured using a dual-carbon-source simultaneous supply method to obtain... 13 C-labeled micro-tuberous roots of Codonopsis pilosula; (4) From the above 13 C-labeled extract from Codonopsis pilosula micro-tuberous roots 13 C-labeled Codonopsis pilosula polysaccharide PF40.

2. The method according to claim 1, characterized in that, The culture medium does not contain sucrose, and the concentration of glucose is 5-45 g / L.

3. The method according to claim 1, characterized in that, The 13 C-labeled glucose is 13 C6-glucose.

4. The method according to claim 1, characterized in that, The 13 C-labeled carbon dioxide through 13 C-NaHCO3 reacts with sulfuric acid to produce the product, which is then introduced into the tissue culture container.

5. The method according to claim 1, characterized in that, The carbon dioxide is replenished once every 1-7 days, and a single replenishment increases the carbon dioxide concentration in the tissue culture container from 400 ppm to 1500 ppm.

6. The method according to claim 1, characterized in that, The sterile carbon dioxide replenishment system includes: (a) a gas generating module for generating carbon dioxide through the reaction of acid and bicarbonate; (b) a gas filtration and delivery module, the outlet of which is connected to the gas filtration and delivery module, the gas filtration and delivery module including at least one 0.22 μm hydrophobic filter membrane unit and sterile tubing; (c) a manifold distribution module connected to the gas filtration and delivery module, the manifold distribution module having multiple branch lines and each branch line having an independent valve; and (d) an end-connection module connected to each branch line for introducing carbon dioxide filtered through the 0.22 μm hydrophobic filter membrane into multiple tissue culture containers.

7. The method according to claim 6, characterized in that, The gas generating module includes a container for holding acid and a syringe interface for adding bicarbonate solution.

8. The method according to claim 6, characterized in that, Each branch of the manifold diversion module is equipped with a 0.22 μm hydrophobic filter membrane unit.

9. The method according to claim 6, characterized in that, Each tissue culture container has a central opening in its cap and a vent connector that connects to the end access module. Carbon dioxide enters the gas phase space of the tissue culture container through the vent connector.

10. The method according to claim 6, characterized in that, Each tissue culture container is equipped with a breathable needle that is connected to a 0.22 μm hydrophobic filter membrane unit, serving as an exhaust valve for the tissue culture container to achieve pressure balance and maintain a sterile barrier.

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