Granulosa cell in vitro culture device based on spermidine release

Abstract

The utility model belongs to the field of biological cell in-vitro culture technology, concretely relates to a kind of granulosa cell in-vitro culture device based on spermidine slow-release, including culture vessel, the bottom of culture vessel is provided with temperature control module, the inner wall of the bottom of culture vessel is provided with spermidine slow-release module, spermidine slow-release module includes porous slow-release material layer and spermidine, and spermidine is arranged in the inside of porous slow-release material layer;The culture device is through the synergistic effect of temperature control module and spermidine slow-release module, realizes the sustained stable release of spermidine and the monitoring control of granulosa cell culture environment, improves granulosa cell activity and functional expression, and has the advantages such as simple operation, low cost, strong compatibility, suitable for large-scale cell culture and drug screening.

Description

Technical Field

[0001] This invention belongs to the field of in vitro culture technology of biological cells, specifically relating to an in vitro culture device for granular cells based on spermidine sustained release. Background Technology

[0002] Granulosa cells are the main functional components of the follicular microenvironment, and their in vitro expansion and maintenance of activity are of great significance for reproductive medicine research, the construction of endocrine disease models, and assisted reproductive technologies. However, once granulosa cells are removed from the in vivo environment, they are prone to functional decline. The core reasons include a sharp increase in oxidative stress levels, unstable supply of key active factors, and difficulty in controlling culture conditions.

[0003] Spermine is an important polyamine for maintaining granulocyte homeostasis. It can significantly enhance cell activity and steroid hormone secretion by inducing autophagy to clear damaged mitochondria, stabilizing mitochondrial membrane potential, and reducing the expression of inflammatory factors. However, spermine requires repeated artificial addition during conventional culture, which cannot achieve stable and continuous release of spermine during culture. This leads to drastic fluctuations in concentration, which can easily cause cellular metabolic disorders and thus affect normal cell growth and functional expression.

[0004] Meanwhile, existing culture devices generally lack dynamic temperature control modules, which cannot provide a suitable and stable temperature environment for granulocytes. Temperature fluctuations will adversely affect cell metabolism and function, resulting in a cell survival rate of less than 60%, which seriously restricts the effect and quality of granulocyte in vitro culture and limits its application in drug screening, reproductive medicine research and other fields. Utility Model Content

[0005] To address the aforementioned shortcomings of existing technologies, this invention provides a granulocyte in vitro culture device based on spermidine sustained release, which solves the problem that existing granulocyte in vitro culture devices cannot stably and continuously release spermidine.

[0006] To achieve the above objectives, the technical solution adopted by this utility model is as follows: A spermidine-based in vitro culture device for granulocytes is provided, comprising a culture container, a temperature control module at the bottom of the culture container, and a spermidine-based sustained-release module on the inner wall of the bottom of the culture container. The spermidine-based sustained-release module includes a porous sustained-release material layer and spermidine, with the spermidine disposed inside the porous sustained-release material layer.

[0007] The beneficial effects of adopting the above technical solution are as follows: This culture device, through the synergistic effect of the sustained-release module and the temperature control module, achieves continuous and stable release of spermidine and precise control of the culture environment, thereby solving the problem of cell metabolic disorders caused by large fluctuations in spermidine concentration in traditional culture methods. Specifically, the temperature control module can monitor and control the culture environment inside the culture container, avoiding cell damage caused by fluctuations or abnormalities in environmental conditions, and improving the stability and success rate of granulosa cell culture; the spermidine sustained-release module uses a porous sustained-release material layer to load spermidine, achieving long-term sustained release of spermidine, avoiding the need for frequent addition of spermidine, ensuring the continuous stability of spermidine concentration in the cell environment, and thus maintaining the stability of granulosa cell activation and functional expression.

[0008] Furthermore, the porous sustained-release material layer includes a polylactic acid-glycolic acid copolymer membrane and sodium alginate-chitosan composite microspheres arranged sequentially from top to bottom.

[0009] The beneficial effects of the above technical solution are as follows: The porous sustained-release material layer adopts a double-layer structure with an inner layer of sodium alginate-chitosan composite microspheres and an outer layer of polylactic acid-glycolic acid copolymer membrane (PLGA). This ensures the persistence and stability of spermidine sustained release and provides a safe and controllable culture environment for granulocytes, thereby improving the in vitro culture effect of granulocytes. Among them, the polylactic acid-glycolic acid copolymer membrane (PLGA) serves as the outer barrier of the porous sustained-release material layer, which can effectively control the release rate of spermidine and prevent external contamination. The sodium alginate-chitosan composite microspheres serve as the inner layer structure, which can provide loading space for spermidine, achieving efficient loading and long-term sustained release of spermidine. At the same time, the sodium alginate-chitosan composite microspheres can promote cell adhesion and growth.

[0010] Furthermore, the spermidine sustained-release module is arranged in a grid pattern at the bottom of the culture container.

[0011] The beneficial effects of adopting the above technical solution are as follows: the spermidine sustained-release module is set in a grid shape at the bottom of the culture container, which can increase the contact area with the culture medium, improve the uniformity of spermidine release and diffusion efficiency, and avoid cell metabolic disorders caused by excessively high or low local concentrations.

[0012] Furthermore, the bottom of the culture container is provided with a groove, and the spermidine sustained-release module is disposed in the groove.

[0013] The beneficial effects of adopting the above technical solution are as follows: the groove at the bottom of the culture container can provide a fixed position for the spermidine sustained-release module, preventing displacement or detachment caused by the flow or vibration of the culture medium during the culture process, thus ensuring the stability of the spermidine sustained-release module; at the same time, the groove increases the contact area between the spermidine sustained-release module and the culture medium, promoting the uniform diffusion of spermidine and avoiding cell metabolic disorders caused by excessively high or low local concentrations, thereby improving the culture effect and survival rate of granulocytes.

[0014] Furthermore, the temperature control module includes a temperature sensor and a heating element, with the heating element positioned on the outer circumferential surface of the culture container.

[0015] The beneficial effects of adopting the above technical solution are as follows: the temperature control module, through a temperature sensor and a heating element set on the outer circumference of the culture container, realizes the control of the temperature in the culture environment, ensures the constantness of the culture environment, effectively reduces the impact of temperature fluctuations on the activity of granulocytes, and thus improves the culture success rate.

[0016] Furthermore, the heating element is a ring-shaped silicone heating band.

[0017] The beneficial effects of adopting the above technical solution are as follows: the heating element adopts a ring-shaped silicone heating band, which can be closely attached to the outer circumference of the culture container, ensuring the uniformity of heating of the culture container, avoiding the problems of local overheating or uneven temperature, and providing a stable and suitable heating environment for granulocytes, thereby improving the success rate of granulocyte culture.

[0018] Furthermore, the temperature control module also includes a humidity sensor, which is located inside the culture container.

[0019] The beneficial effects of adopting the above technical solution are as follows: the humidity sensor installed inside the culture container can monitor the humidity changes of the culture cell environment in real time, avoiding the risk of microbial contamination caused by excessively low humidity or excessively high humidity, thereby maintaining the stability of granulocyte activation and functional expression.

[0020] Furthermore, the top of the culture container is provided with a culture sealing cap, and a gas exchange filter membrane is provided on the culture sealing cap.

[0021] The beneficial effects of adopting the above technical solution are as follows: the culture sealing cap can effectively block external pollutants from entering the culture container, avoiding the risk of cross-contamination; the gas exchange filter membrane can ensure a sterile environment while allowing free exchange of gases such as oxygen and carbon dioxide, maintaining the gas balance and pH stability in the culture medium, which is conducive to the growth of granulocytes, thereby improving the survival rate and functional expression of granulocytes in vitro.

[0022] In summary, the beneficial effects of the spermidine-based granulocyte in vitro culture device provided by this invention are as follows: This culture device achieves continuous and stable release of spermidine and monitoring and control of the granulocyte culture environment through the synergistic effect of a temperature control module and a spermidine sustained-release module. The spermidine sustained-release module adopts a double-layer structure with an inner layer of sodium alginate-chitosan composite microspheres and an outer layer of polylactic acid-glycolic acid copolymer membrane (PLGA). This ensures the persistence and stability of spermidine sustained release and provides a safe and controllable culture environment for granulocytes, thereby improving the in vitro culture effect of granulocytes. The temperature control module maintains a constant culture temperature through the synergistic work of a temperature sensor and a heating element, avoiding the impact of temperature fluctuations on granulocyte activity. This significantly improves the survival rate and functional expression of granulocytes, solving the problems of large fluctuations in spermidine concentration and cell metabolic disorders caused by repeated artificial addition in traditional culture methods. Moreover, the entire culture device is simple to operate, low in cost, and suitable for large-scale cell culture and drug screening. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the overall structure of this utility model; Figure 2 This is a top view of the present invention; Figure 3 This is a schematic diagram of the spermidine sustained-release module in this utility model; Figure 4 Top view of the spermidine sustained-release module in my utility model; Figure 5 This is a connection diagram of the temperature control module in this utility model; The components include: 1. Culture container; 2. Spermine sustained-release module; 21. Porous sustained-release material layer; 22. Spermine; 3. Temperature control module; 31. Temperature sensor; 32. Heating element; 33. Humidity sensor; and 4. Gas exchange filter membrane. Detailed Implementation

[0024] The specific embodiments of this utility model are described below to enable those skilled in the art to understand this utility model. However, it should be understood that this utility model is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of this utility model as defined and determined by the appended claims, these changes are obvious. All utility model creations utilizing the concept of this utility model are within the scope of protection.

[0025] like Figures 1-5As shown, the granulocyte in vitro culture device based on spermidine sustained release provided by this utility model includes a culture container 1, a temperature control module 3 at the bottom of the culture container 1, and a groove for fixing a spermidine sustained release module 2 at the bottom of the culture container 1. The spermidine sustained release module 2 is arranged in a grid pattern at the bottom of the culture container 1 and includes a porous sustained release material layer 21 and spermidine 22, with spermidine 22 disposed inside the porous sustained release material layer 21. This culture device, through the synergistic effect of the spermidine sustained release module 2 and the temperature control module 3, achieves continuous and stable release of spermidine 22 and precise control of the culture environment, thereby solving the problem of cell metabolic disorders caused by large fluctuations in spermidine 22 concentration in traditional culture methods. Among them, the temperature control module 3 can monitor and control the culture environment inside the culture container 1, avoid cell damage caused by fluctuations or abnormalities in environmental conditions, and improve the stability and success rate of granulocyte culture; the spermidine sustained release module 2 uses a porous sustained release material layer 21 to load spermidine 22, realize the long-term sustained release of spermidine 22, avoid the need for frequent addition of spermidine 22, ensure the continuous stability of spermidine 22 concentration in the cell environment, and thus maintain the stability of granulocyte activation and functional expression.

[0026] like Figure 3 and Figure 4 As shown, the porous sustained-release material layer 21 includes a polylactic acid-glycolic acid copolymer membrane (PLGA) and sodium alginate-chitosan composite microspheres arranged sequentially from top to bottom. The thickness of the polylactic acid-glycolic acid copolymer membrane (PLGA) is 0.4-0.5 mm, the pore size of the sodium alginate-chitosan composite microspheres is 50-200 μm, and the spermidine 22 loading is 15 mg / cm². The porous sustained-release material layer 21 adopts a double-layer structure with an inner layer of sodium alginate-chitosan composite microspheres and an outer layer of polylactic acid-glycolic acid copolymer membrane (PLGA), which not only ensures the persistence and stability of the sustained release of spermidine 22, but also provides a safe and controllable culture environment for granulocytes, thereby improving the in vitro culture effect of granulocytes. In use, spermidine 22 is loaded into the porous structure of the porous sustained-release material layer 21. The pore size of the sodium alginate-chitosan composite microspheres (50-200 μm) restricts molecular diffusion, thereby allowing spermidine 22 to be released slowly and continuously from the porous structure of the porous sustained-release material layer 21 into the culture medium. This avoids drastic fluctuations in the concentration of spermidine 22. Furthermore, the polylactic acid-glycolic acid copolymer membrane (PLGA) can further control the release rate, ensuring the stable release of spermidine 22 and preventing external contamination, thus maintaining the activity and functional stability of granulocytes.

[0027] like Figure 1 and Figure 5As shown, the temperature control module 3 includes a temperature sensor 31 and a heating element 32. The heating element 32 is set on the outer circumferential surface of the culture container 1 by a high-temperature resistant adhesive, and the heating element 32 is a ring-shaped silicone heating strip. Both the temperature sensor 31 and the heating element 32 are electrically connected to a PID controller, which is used to control the working state of the heating element 32 according to the feedback signal of the temperature sensor 31, so as to control the temperature in the culture environment and maintain it at 37℃±0.5℃, ensuring the constantness of the granulocyte culture environment, thereby effectively reducing the impact of temperature fluctuations on the activity of granulocytes and improving the culture effect and survival rate of granulocytes.

[0028] like Figure 5 As shown, the temperature control module 3 also includes a humidity sensor 33, which is electrically connected to the PID controller. The humidity sensor 33 is located inside the culture container 1. The humidity sensor 33 can monitor the humidity changes of the culture granulocyte environment in real time, avoiding the risk of microbial contamination caused by excessively low humidity or excessively high humidity, thereby maintaining the stability of granulocyte activation and functional expression.

[0029] like Figure 1 As shown, the top of the culture container 1 is equipped with a culture sealing cap, and a gas exchange filter membrane 4 is installed on the culture sealing cap. The gas exchange filter membrane 4 is made of polytetrafluoroethylene (PTFE) material and has a thickness of 0.22 μm. The culture sealing cap can effectively block external contaminants from entering the culture container 1, avoiding the risk of cross-contamination. The gas exchange filter membrane 4 can ensure a sterile environment while allowing free exchange of gases such as oxygen and carbon dioxide, maintaining the gas balance and pH stability in the culture medium, which is conducive to the growth of granulocytes, thereby improving the survival rate and functional expression of granulocytes in vitro.

[0030] In summary, the spermidine-based granulocyte culture device provided by this invention achieves continuous and stable release of spermidine 22 and monitoring and control of the granulocyte culture environment through the synergistic effect of the temperature control module 3 and the spermidine sustained-release module 2. The spermidine sustained-release module 2 employs a double-layer structure, with an inner layer of sodium alginate-chitosan composite microspheres providing a high loading space for spermidine 22, and an outer polylactic acid-glycolic acid copolymer membrane (PLGA) forming a physical barrier. Spermine 22 first slowly diffuses through the pores of the sodium alginate-chitosan composite microspheres. The release rate of spermidine 22 is controlled by a polylactic-co-glycolic acid (PLGA) membrane, ensuring the sustained and stable release of spermidine 22. At the same time, the temperature control module 3 maintains a constant temperature environment in the culture device through the synergistic effect of the temperature sensor 31 and the heating element 32. This ensures both the activity of granulocytes and the constant degradation rate of the polylactic-co-glycolic acid (PLGA) membrane, avoiding the concentration fluctuations caused by repeated artificial addition of spermidine 22 during conventional culture, thereby maintaining the stability of granulocyte activation and functional expression.

Claims

1. A device for in vitro culture of granulosa cells based on spermidine sustained release, characterized in that: The system includes a culture container (1), a temperature control module (3) is provided at the bottom of the culture container (1), and a spermidine sustained-release module (2) is provided on the inner wall of the bottom of the culture container (1). The spermidine sustained-release module (2) includes a porous sustained-release material layer (21) and spermidine (22), and the spermidine (22) is disposed inside the porous sustained-release material layer (21).

2. The granulosa cell in vitro culture device based on spermidine sustained release according to claim 1, characterized in that: The porous sustained-release material layer (21) includes a polylactic acid-glycolic acid copolymer membrane and sodium alginate-chitosan composite microspheres arranged sequentially from top to bottom.

3. The granulocyte in vitro culture device based on spermidine sustained release according to claim 1, characterized in that: The spermidine sustained-release module (2) is arranged in a grid pattern at the bottom of the culture container (1).

4. The granulocyte in vitro culture device based on spermidine sustained release according to claim 1, characterized in that: The bottom of the culture container (1) is provided with a groove, and the spermidine sustained-release module (2) is disposed in the groove.

5. The granulocyte in vitro culture device based on spermidine sustained release according to claim 1, characterized in that: The temperature control module (3) includes a temperature sensor (31) and a heating element (32), the heating element (32) being disposed on the outer circumferential surface of the culture container (1).

6. The granulocyte in vitro culture device based on spermidine sustained release according to claim 5, characterized in that: The heating element (32) is an annular silicone heating band.

7. The granulosa cell in vitro culture device based on spermidine sustained release according to claim 5, characterized in that: The temperature control module (3) also includes a humidity sensor (33), which is located inside the culture container (1).

8. The granulocyte in vitro culture device based on spermidine sustained release according to claim 1, characterized in that: The top of the culture container (1) is provided with a culture sealing cap, and a gas exchange filter membrane (4) is provided on the culture sealing cap.

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