Carbon dioxide incubator and culture method thereof

Abstract

The invention discloses a carbon dioxide incubator and a culture method thereof, and relates to the technical field of incubators, the incubator comprises an incubator body, a glass door is hinged to the front side of the incubator body, and a fan switch is installed on the front side of the glass door; the temperature control system is combined with the PID control algorithm, the temperature in the incubator is accurately controlled within the range of the set value + / -0.5 DEG C, the influence of temperature fluctuation on cell growth is avoided, the stability of cell metabolism and growth is guaranteed, the humidity is stably maintained at the set value + / -2% through the humidity control system, a culture medium is effectively prevented from being evaporated, and the cell quality is improved. The high-humidity environment required by cell growth is maintained, the concentration of carbon dioxide is stabilized at about 5% through a carbon dioxide control system, the pH of a culture medium is stable, appropriate conditions are provided for normal physiological activities of cells, in addition, the incubator is easy and convenient to operate and high in function integration degree, diversified experiment requirements are met, and popularization and application are facilitated. The effects of improving the experiment reliability, repeatability and experiment efficiency are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of incubators, and particularly to a carbon dioxide incubator and a culture method thereof. Background Art

[0002] A carbon dioxide incubator is a closed cell culture device, and its core function is to simulate the growth environment of cells in vivo, providing stable conditions for the in vitro culture and research of cells and tissues. In many fields such as biomedical engineering, cell biology, and tissue engineering, the in vitro culture of cells and tissues is an important means to deeply study life phenomena, disease mechanisms, and develop treatment methods. The cell growth environment in the human body or animal body has specific temperature, humidity, and CO 2 concentration. Cells can only metabolize, grow, and differentiate normally in such an environment. For example, the normal body temperature of the human body is about 37 °C, the humidity of extracellular fluid is relatively high, and the CO 2 concentration is within a certain range, maintaining a stable acid-base balance. The carbon dioxide incubator is precisely designed to reproduce these conditions in vitro so that cells can grow in an environment similar to that in vivo, thereby ensuring the accuracy and reliability of experimental results.

[0003] In practical applications, there are many technical problems in existing carbon dioxide incubators. In terms of temperature control, the temperature accuracy of existing incubators is not high enough, and it is difficult to stably maintain the set temperature. In terms of humidity control, existing incubators cannot effectively maintain a high-humidity environment, and the culture medium is prone to evaporation, resulting in a change in the concentration of nutrient components, which in turn affects the normal physiological functions of cells. The CO 2 concentration adjustment of existing incubators is not precise enough, and it cannot be stably maintained at the appropriate level required for cell growth, resulting in a large fluctuation in the pH value of the culture medium. Existing incubators also have deficiencies in function integration and operation convenience, unable to meet the diverse needs of different experiments, with complex operations and prone to errors. These problems limit the application effect of carbon dioxide incubators in scientific research and production. Therefore, it is of great significance to develop a carbon dioxide incubator and a culture method thereof. Summary of the Invention

[0004] The objective of the present invention is to make up for the deficiencies of the prior art and provide a carbon dioxide incubator and its cultivation method. It can accurately control the temperature inside the incubator within the range of the set value ±0.5°C, avoid the influence of temperature fluctuations on cell growth, ensure the stability of cell metabolism and growth, stably maintain the humidity within the range of the set value ±2%, effectively prevent the evaporation of the culture medium, maintain the high-humidity environment required for cell growth, can stably maintain the carbon dioxide concentration at about 5%, ensure that the pH of the culture medium is stable at 7.2 - 7.4, provide suitable conditions for the normal physiological activities of cells, and the incubator is easy to operate and has a high degree of functional integration, meeting diverse experimental needs and achieving the effects of improving experimental reliability, repeatability, and experimental efficiency.

[0005] To solve the above technical problems, the present invention provides the following technical solution: A carbon dioxide incubator, which includes a box body. A glass door is hinged to the front of the box body. A fan switch is installed on the front of the glass door. A door latch is installed on the front of the glass door. A control panel is installed on the front of the box body. Two shelves are installed on the inner wall of the box body. A box door is hinged to the front of the box body. A temperature control system, a humidity control system, and a carbon dioxide control system are installed inside the box body.

[0006] Further, the temperature control system consists of a temperature controller, a temperature sensor, and a heater, and is used to keep the temperature inside the box body of the incubator within the set temperature range, achieving precise control of the temperature inside the box.

[0007] Even further, the humidity control system consists of a humidity controller, a humidity sensor, and a humidifier, and is used to keep the humidity inside the box body at the set level, maintaining a high-humidity environment inside the box to prevent the evaporation of the culture medium.

[0008] Even further, the carbon dioxide control system is the core part of the carbon dioxide incubator. Its main function is to control and regulate carbon dioxide through molecular sieve adsorption method, chemical absorption method, and membrane separation method technologies. The carbon dioxide control system consists of a carbon dioxide sensor, a carbon dioxide controller, and a carbon dioxide gas source, and is used to adjust the input of carbon dioxide gas and maintain the pH stability of the culture medium.

[0009] A cultivation method for a carbon dioxide incubator, applicable to the above carbon dioxide incubator, the method includes the following steps: Preparation stage: Connect the power supply of the incubator, start the temperature control system, humidity control system, and carbon dioxide control system through the control panel for self-check. According to the experimental requirements, set the temperature control system to the target temperature, the humidity control system to the target humidity, and the carbon dioxide control system to the target carbon dioxide concentration. At the same time, check the installation of the shelves inside the box body and adjust their positions according to the size of the culture container; Culture medium preparation: Select a culture medium according to the types of cultured cells and tissues, add nutrients and serum according to the instructions, and treat the culture medium using standard sterilization methods such as filtration sterilization and autoclaving to ensure its aseptic state; Sample placement: Open the chamber door and the glass door. Under aseptic operating conditions, place the culture containers containing cells and tissues on the adjusted shelves. After placement, quickly close the glass door and the chamber door to reduce the gas exchange between the internal environment of the chamber and the outside.

[0010] Further, when the temperature control system is working, the temperature sensor continuously collects the temperature data inside the incubator , and the temperature controller calculates the difference between the set temperature and the collected temperature , , and calculates and outputs the control quantity according to the PID control algorithm, and then adjusts the heating power of the heater . The formula of the PID control algorithm is: , where is the proportional coefficient, is the integral time constant, is the differential time constant. Through this algorithm, the temperature inside the incubator is stabilized within ±0.5 °C of the set temperature to maintain the suitable temperature environment required for cell growth.

[0011] Even further, when the humidity control system is running, the humidity sensor continuously monitors the humidity data inside the chamber , and the humidity controller compares it with the set humidity to obtain the humidity difference , . When , it indicates that the humidity inside the chamber is lower than the set value, and the humidifier starts. Its humidification amount is calculated according to the following formula: , where , are coefficients related to the performance of the humidifier. By adjusting the humidification amount, the humidity inside the chamber is stably maintained within ±2% of the set humidity to maintain the high humidity environment required for cell growth.

[0012] Even further, when the carbon dioxide control system is running, the carbon dioxide sensor continuously detects the carbon dioxide concentration data inside the chamber , and the carbon dioxide controller compares it with the set concentration to obtain the concentration difference , . When When the carbon dioxide gas source is turned on to supplement carbon dioxide, its supplementary flow rate is calculated according to the following formula: , where , are coefficients related to the performance of the carbon dioxide control system. The carbon dioxide concentration is adjusted using this system and formula to maintain the pH of the culture medium stable within a range suitable for cell growth.

[0013] Furthermore, during the entire culture process, at regular time intervals, the growth status of cells and tissues is observed through the glass door of the incubator. If it is found that the color of the culture medium in the culture container has changed significantly, it means that the pH value has changed. At this time, the real-time data of carbon dioxide concentration, temperature, and humidity need to be viewed through the control panel, and the necessary adjustments are made to each system parameter to maintain a stable culture environment. After the culture is completed, first turn off the carbon dioxide gas source, and then turn off the temperature control system, humidity control system, and power supply in sequence. Take out the culture container, wipe and disinfect the inner wall, shelf, and glass door of the incubator with 75% alcohol, and then irradiate with an ultraviolet lamp for 30 - 60 minutes for sterilization treatment to prepare for the next use.

[0014] Compared with the prior art, the carbon dioxide incubator and its culture method have the following beneficial effects: First, through the temperature control system combined with the PID control algorithm, the present invention can accurately control the temperature in the incubator within the range of ±0.5°C of the set value, avoiding the influence of temperature fluctuations on cell growth, and ensuring the stability of cell metabolism and growth. Through the humidity control system, the humidity is stably maintained within ±2% of the set value, effectively preventing the evaporation of the culture medium and maintaining the high-humidity environment required for cell growth. Through the carbon dioxide control system, the carbon dioxide concentration is stably maintained at about 5%, ensuring the stability of the pH of the culture medium and providing suitable conditions for the normal physiological activities of cells. In addition, the incubator is easy to operate and has a high degree of functional integration, meeting the diverse experimental needs and achieving the effects of improving experimental reliability, repeatability, and experimental efficiency.

[0015] Second, through the stable culture environment, the differences in the growth environments of cells and tissues in different batches of experiments are significantly reduced. Cells under multiple experimental conditions grow in the same stable environment, and their differences can more accurately reflect the effects of experimental treatments rather than errors caused by environmental factors, enhancing the repeatability of the experiment. The supporting culture method details a series of operation steps from preparation work, culture medium preparation, sample placement to culture process monitoring, culture medium replacement, subculture, and cleaning and disinfection after the culture is completed, enabling experimental personnel to use the incubator in a standardized and convenient manner and reducing interference with the experiment caused by improper operation.

[0016] Other advantages, objects and features of the present invention will be set forth in part in the following description, and in part will be obvious to those skilled in the art upon examination of the following, or may be learned from the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention, and for those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is a schematic three-dimensional structure diagram of a carbon dioxide incubator; Figure 2 It is a flowchart of a cultivation method of a carbon dioxide incubator.

[0019] In the figure: 1, the box body; 2, the fan switch; 3, the door roller; 4, the glass door; 5, the control panel; 6, the shelf; 7, the box door. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following will, in conjunction with the accompanying drawings and preferred embodiments, describe in detail the specific embodiments, structures, features and their effects of the present invention as follows.

[0021] Embodiment 1 This embodiment elaborates in detail the structure, connection relationship, functions and working principles of the carbon dioxide incubator. The carbon dioxide incubator mainly consists of a box body 1, a glass door 4, a fan switch 2, a door roller 3, a control panel 5, a shelf 6, a box door 7, as well as a temperature control system, a humidity control system and a carbon dioxide control system.

[0022] The box body 1 serves as the main body of the incubator, providing physical support and space for the internal components and the cultivation environment. The glass door 4 is hinged to the front of the box body 1, facilitating the experimenter to observe the situation inside the box, and at the same time effectively reducing the heat exchange and gas exchange between the internal environment of the box and the outside. The fan switch 2 and the door roller 3 are installed on the front of the glass door 4. The fan switch 2 is used to control the air circulation fan inside the box, promoting the uniform distribution of temperature, humidity and gas concentration inside the box.

[0023] The door clamp 3 is used to fix the glass door 4 to ensure good sealing when it is closed. The control panel 5 is installed on the front of the cabinet 1 and is the key interface for users to interact with the incubator. Users can set parameters such as temperature, humidity, and carbon dioxide concentration here and view the working status of each system in real time. The two shelves 6 on the inner wall of the cabinet 1 are used to place culture containers such as culture bottles and petri dishes. The cabinet door 7 is also hinged on the front of the cabinet 1 to further enhance the sealing of the incubator and reduce the interference of the external environment on the culture environment inside the box.

[0024] The temperature control system consists of a temperature controller, a temperature sensor, and a heater. Its working principle is based on precise temperature monitoring and control algorithms. When working, the temperature sensor continuously collects the temperature data inside the incubator , and the temperature controller compares the set temperature with the collected temperature to obtain the difference , .

[0025] The temperature controller calculates and outputs the control quantity according to the PID control algorithm, and then adjusts the heating power of the heater . The formula of the PID control algorithm is: , where is the proportional coefficient, is the integral time constant, is the differential time constant. For example, when the set temperature is 37 °C, if the temperature sensor detects that the temperature inside the box is lower than 37 °C, a positive is generated. The temperature controller calculates the required increased heating amount according to the PID algorithm and controls the heater to increase the heating power; conversely, if is higher than 37 °C, is negative, and the temperature controller controls the heater to reduce the heating power or stop heating, so as to accurately control the temperature inside the incubator within the range of ±0.5 °C of the set temperature to create a stable temperature environment for cell growth.

[0026] The humidity control system consists of a humidity controller, a humidity sensor, and a humidifier. The humidity sensor continuously monitors the humidity data inside the box , and the humidity controller compares it with the set humidity (usually about 95%) to obtain the humidity difference , , when , it indicates that the humidity inside the box is lower than the set value, and the humidifier starts. Its humidification amount is calculated according to the formula , where , is a coefficient related to the performance of the humidifier.

[0027] The humidifier increases the humidity inside the chamber by means of water pan evaporation or direct injection of water vapor. When the humidity reaches the set value, the humidifier stops working, thereby stably maintaining the humidity inside the chamber 1 within ±2% of the set humidity to effectively prevent the evaporation of the culture medium and maintain the high humidity environment required for cell growth.

[0028] The carbon dioxide control system is the core part of the carbon dioxide incubator, which consists of a carbon dioxide sensor, a carbon dioxide controller, and a carbon dioxide gas source. It mainly realizes the precise control and regulation of carbon dioxide by means of molecular sieve adsorption method, chemical absorption method, and membrane separation method. The carbon dioxide sensor continuously detects the carbon dioxide concentration data inside the chamber and the carbon dioxide controller compares it with the set concentration (generally about 5%) to obtain the concentration difference , .

[0029] When , the carbon dioxide gas source is turned on to supplement carbon dioxide, and its supplementary flow rate is calculated according to the formula . Among them, , are coefficients related to the performance of the carbon dioxide control system. By adjusting the carbon dioxide concentration through this system and formula, the pH of the culture medium is stably maintained within the range suitable for cell growth, providing a stable acid-base environment for the cells.

[0030] Example 2 This example fully presents the culture method of the carbon dioxide incubator, covering the entire process of operation from preparation to after cultivation.

[0031] Before using the carbon dioxide incubator, preparatory work needs to be done. Connect the power supply of the incubator, the control panel 5 lights up, and each system automatically starts self-checking. Start the temperature control system, humidity control system, and carbon dioxide control system in sequence through the control panel 5, and closely observe the status indicators of each system on the control panel to ensure that all systems are operating normally.

[0032] According to the type of cells or tissues to be cultured in the experiment, select a suitable culture medium. For example, when culturing mammalian cells, select the corresponding cell culture medium, and strictly add various nutrients, serum and other substances according to the instructions. After preparation, the culture medium is sterilized by filtration using a 0.22 μm filter membrane to ensure that the culture medium is sterile.

[0033] Check the installation stability of the shelf 6 inside the box body 1. According to the size and quantity of the culture containers, reasonably adjust the position of the shelf 6. Open the box door 7 and the glass door 4. On the sterile workbench, carefully place the culture containers containing cells or tissues on the shelf 6 with tweezers or a pipette, paying attention to maintaining balance, avoiding shaking and collision, and preventing damage to the cells or tissues. After placing, quickly close the glass door 4 and the box door 7 to reduce the gas exchange and temperature change between the internal environment of the box and the outside world.

[0034] Set the culture parameters through the control panel, set the temperature to 37°C, the humidity to 95%, and the carbon dioxide concentration to 5%. At this time, the temperature control system, humidity control system, and carbon dioxide control system start to adjust the internal environment of the box according to their respective working principles and calculation formulas.

[0035] During the culture process, every 2 - 3 hours, observe the growth state of the cells or tissues in the culture containers through the glass door 4, such as whether the cell morphology is normal, whether there are signs of proliferation or differentiation, etc. At the same time, pay attention to the color change of the culture medium. If the culture medium turns yellow, it may mean that the acidic substances produced by cell metabolism increase, the carbon dioxide concentration is too high, or the pH value drops; if it turns red, it may be that the carbon dioxide concentration is too low or the pH value rises. Once an abnormality is found, immediately check the real-time data of temperature, humidity, and carbon dioxide concentration through the control panel 5.

[0036] Taking temperature as an example, if the temperature is found to be abnormal, judge whether adjustment is needed according to the PID control algorithm of the temperature control system. The algorithm formula is: )). If the temperature deviates from the set value, the relevant coefficients can be manually fine-tuned 、 、 to ensure that the temperature is stable within the range of ±0.5°C of the set temperature. For humidity and carbon dioxide concentration, if abnormalities occur, also according to their respective calculation formulas, the humidification amount Calculation formula: ; the carbon dioxide supplement flow rate Calculation formula: , combined with the actual situation, such as appropriately adjusting the working frequency of the humidifier or the carbon dioxide gas source supplement flow rate, to ensure that the humidity is within the range of ±2% of the set humidity, and the carbon dioxide concentration is maintained around the set value, ensuring the stability of the pH of the culture medium.

[0037] If it is necessary to add or replace the culture medium, first open the glass door 4. Under sterile conditions, carefully suck out the old culture medium in the culture container with a sterile pipette as much as possible, and then slowly add the pre-prepared fresh culture medium, paying attention to avoiding the culture medium from contacting the mouth or wall of the culture container to prevent contamination. After adding or replacing the culture medium, immediately close the glass door 4 to reduce the fluctuation of the internal environment of the box.

[0038] When the cultured cells or tissues reach a specific growth stage and subculture is required, first take out the culture containers in the incubator and place them on the sterile operating table. According to the standard operating procedures for cell subculture, use digestive fluids such as trypsin to digest the cells from the surface of the culture containers to make a cell suspension. Then, appropriately dilute the cell suspension and aliquot it into new culture containers. Put the new culture containers back into the incubator, reset the culture parameters, and continue the culture.

[0039] After the culture is completed, first turn off the carbon dioxide gas source to stop supplying carbon dioxide into the chamber. Subsequently, turn off the temperature control system and the humidity control system. Finally, turn off the power supply, take out the culture containers, and clean and disinfect the incubator. First, wipe the inner wall of the chamber 1, the shelf 6, the glass door 4, etc. with a damp cloth to remove surface stains and residual culture medium. Then, wipe the above-mentioned parts with 75% alcohol for disinfection. Finally, open the chamber door 7 and the glass door 4, and irradiate the incubator with an ultraviolet lamp for 30 - 60 minutes to thoroughly sterilize it and make preparations for the next use.

[0040] The above are only the preferred embodiments of the present invention and do not impose any formal limitations on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A carbon dioxide incubator, characterized in that: The incubator comprises a box body (1), a glass door (4) is hingedly connected to the front of the box body (1), a fan switch (2) is installed on the front of the glass door (4), a door rolling head (3) is installed on the front of the glass door (4), a control panel (5) is installed on the front of the box body (1), two shelves (6) are installed on the inner wall of the box body (1), a box door (7) is hingedly connected to the front of the box body (1), and a temperature control system, a humidity control system and a carbon dioxide control system are installed inside the box body (1).

2. A carbon dioxide incubator according to claim 1, characterized in that: The temperature control system is composed of a temperature controller, a temperature sensor, and a heater, and is used to maintain the temperature inside the incubator body (1) within a set temperature range, thereby achieving precise control of the temperature inside the body (1).

3. A carbon dioxide incubator according to claim 1, characterized in that: The humidity control system is composed of a humidity controller, a humidity sensor, and a humidifier, and is used to keep the humidity inside the box (1) at a set level, and is used to maintain a high humidity environment inside the box (1).

4. A carbon dioxide incubator according to claim 1, characterized in that: The carbon dioxide control system is the core part of the carbon dioxide incubator. Its main function is to control and regulate carbon dioxide through molecular sieve adsorption, chemical absorption and membrane separation techniques. The carbon dioxide control system consists of a carbon dioxide sensor, a carbon dioxide controller and a carbon dioxide gas source, and is used to regulate the input of carbon dioxide gas and maintain the pH stability of the culture medium.

5. A cultivation method for a carbon dioxide incubator, applicable to the carbon dioxide incubator according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: Preparation stage: Turn on the power of the incubator, start the temperature control system, humidity control system and carbon dioxide control system through the control panel (5) to perform self-check, and according to the experimental requirements, set the temperature control system to the target temperature, the humidity control system to the target humidity, and the carbon dioxide control system to the target carbon dioxide concentration. At the same time, check the installation of the shelf (6) in the box (1) and adjust its position according to the size of the culture container; Preparation of culture medium: Select culture medium according to the type of cultured cells and tissues, add nutrients and serum according to the instructions, and treat the culture medium by standard sterilization methods such as filtration sterilization and high-pressure steam sterilization to ensure its sterility; Sample placement: Open the box door (7) and the glass door (4), and under aseptic operating conditions, place the culture container containing cells and tissues on the adjusted shelf (6). After placement, quickly close the glass door (4) and the box door (7) to reduce the gas exchange between the environment inside the box (1) and the outside.

6. The cultivation method of a carbon dioxide incubator according to claim 5, characterized in that: When the temperature control system is working, the temperature sensor collects the temperature data in the incubator in real time. , the temperature controller sets the temperature With the collection temperature The difference , , calculate the output control quantity according to the PID control algorithm, and then adjust the heating power of the heater , the PID control algorithm formula is: ,in, is the proportionality coefficient, is the integration time constant, is the differential time constant. Through this algorithm, the temperature in the incubator is stabilized at the set temperature. The temperature range is ±0.5℃, maintaining the appropriate temperature environment for cell growth.

7. The cultivation method of a carbon dioxide incubator according to claim 5, characterized in that: When the humidity control system is running, the humidity sensor monitors the humidity data in the box in real time. , the humidity controller compares it with the set humidity Compare and get the humidity difference , ,when When the humidity in the box (1) is lower than the set value, the humidifier starts and its humidification capacity Calculated according to the following formula: ,in, , is a coefficient related to the performance of the humidifier. By adjusting the humidification amount, the humidity in the box (1) is kept stable at the set humidity. The humidity is kept within the range of ±2% to maintain the high humidity environment required for cell growth.

8. The cultivation method of a carbon dioxide incubator according to claim 5, characterized in that: When the carbon dioxide control system is in operation, the carbon dioxide sensor detects the carbon dioxide concentration data in the box (1) in real time. , the carbon dioxide controller compares it with the set concentration Contrast and get the concentration difference , ,when When the carbon dioxide gas source is turned on to supplement carbon dioxide, the supplementary flow rate Calculated according to the following formula: ,in, , is a coefficient related to the performance of the carbon dioxide control system. The system and formula are used to adjust the carbon dioxide concentration to maintain the pH of the culture medium stable within a range suitable for cell growth.

9. The cultivation method of a carbon dioxide incubator according to claim 5, characterized in that: During the entire culture process, at regular intervals, the growth status of cells and tissues is observed through the glass door (4) of the incubator. If the color of the culture medium in the culture container changes significantly, it means that the pH value has changed. At this time, it is necessary to check the real-time data of carbon dioxide concentration, temperature, and humidity through the control panel, and make necessary adjustments to the parameters of each system to maintain a stable culture environment. After the culture is completed, the carbon dioxide gas source is first turned off, and then the temperature control system, humidity control system and power supply are turned off in turn. The culture container is taken out, and the inner wall of the box (1), the shelf (6), and the glass door (4) are wiped and disinfected with 75% alcohol, and then sterilized with ultraviolet light for 30-60 minutes to prepare for the next use.

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