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The CO2 incubator is a device that simulates the formation of a growth environment similar to that of cells/tissues in an organism. It is a device for cells, and tissues, An advanced instrument for bacterial culture. The incubator requires a stable temperature (typically 37 ° C), a stable CO2 level (typically 5%), a constant pH (naturally 7.2-7.4), and a high relative saturation humidity (95%) to culture cells/tissues in vitro.

Among them, the control of carbon dioxide is the difference between a CO2 incubator and other incubators. The concentration of CO2 is closely related to the pH value of the culture medium. Generally speaking, under a gas environment of 5% for cell culture, cells can maintain a relatively constant pH of 7.2-7.4, especially for cultivating animal cells, which should not exceed 6.8-7.6 in a slightly alkaline environment.

We also designed the incubator to control the environment through 1. temperature, 2. CO2 concentration, and 3. humidity.

1. Temperature control

The temperature controller of the carbon dioxide incubator consists of a platinum resistor as a sensor, a digital control circuit, and an LED digital display circuit. When the sensor outputs a resistance signal proportional to the temperature, it is converted into a voltage signal, which is amplified by the circuit, and then sent to the display circuit to display the measured temperature. The other route is to the comparator for comparison with the set value. When there is a deviation between the two, the output power of the thyristor power tube is triggered to generate heat in the heating tube. When the deviation decreases to zero, the heat emitted by the heating tube also decreases until the heating is stopped. The control circuit has overtemperature and water level alarm functions. When the displayed temperature exceeds the set value of 2.0 ℃, the temperature controller sends out an audible and visual alarm signal while cutting off the output and stopping heating from preventing the temperature from continuing to rise; When the water level in the water tank is too high or too low, the water level controller sends out an audible and visual alarm signal, while cutting off the heating output.

Temperature-controlled heating is divided into air jacket heating and water jacket heating. Both heating systems are accurate and reliable, with advantages and disadvantages.

Water jacket heating maintains a constant temperature through an independent water jacket layer surrounding the internal working chamber. Its advantages: Water is an excellent thermal insulation material. When encountering a power outage, the water jacket system can maintain the temperature accuracy and stability of the incubator for a relatively long time, which is beneficial for users with unstable experimental environments (such as power limitations or frequent power outages).

Air jacket heating is the direct heating of the inner box through heaters throughout the air jacket layer of the box, also known as six-sided direct heating. Compared to the water jacket type, the air jacket type has the characteristics of faster heating and faster temperature recovery than the water jacket type incubator, which is particularly beneficial for short-term cultivation and cultivation that requires frequent opening and closing of the door for sampling.

For users, the air jacket design is more straightforward than the water jacket design (the water jacket type requires adding water, emptying and cleaning the water tank, and frequently monitoring the operation of the water tank, as well as potential pollution hazards).

The ability of water jacket heating to maintain temperature is 4-5 times that of air jacket heating. Without opening the door, even if the power is off, the culture in the tank can maintain for 1 hour or more.

2. Humidity control:

The CO2 incubator is equipped with a water tray to generate humidity in the working chamber for the culture. Generally, the relative humidity can reach 95%. Users can put a water tray into the working chamber during cultivation and add an appropriate amount of distilled water to the water tray to allow it to evaporate naturally.

If the temperature of the CO2 incubator is heated by the water and then detected by the temperature sensor inside the incubator to keep the temperature in the incubator constant at the set temperature. The temperature in the chamber is generally set at about 37 ° C ± 0.2 ° C. During the heating process, the heating signal lamp is always on. When the incubator's temperature reaches the set temperature and stabilizes, the heater stops heating, and the signal light goes out.

Humidity in the chamber is an essential but often overlooked factor for cultivation work. Maintaining a sufficient humidity level and having a fast humidity recovery rate (such as after opening and closing the door) can ensure that excessive drying does not cause culture failure. Currently, most carbon dioxide incubators generate moisture through the evaporation of the humidification rack (the relative humidity level generated can reach about 95%, but the humidity recovery rate is prolonged after opening the door). Choose an incubator with a large humidity evaporation area as much as possible. The more extensive the humidity evaporation area, the easier it is to reach the maximum relative saturation humidity and the shorter the time for humidity recovery after opening and closing the door.

3. CO2 concentration control:

The CO2 gas path control device consists of high-concentration CO2 steel cylinders, gas pumps, pressure stabilizing valves, needle valves, electromagnetic valves, flow meters, and gas storage cylinders. (The user will have to provide high-concentration CO2 steel cylinders). When inputting high-concentration CO2 gas under pressure, the flow rate and time of the regulating valve, needle valve, and solenoid valve are controlled to ensure that a certain amount of CO2 gas enters the working chamber, reaching the CO2 concentration value in the automatic control working chamber. This value is easy to observe and understand. During the operation of the CO2 tank, the air is replenished from the gas storage bottle in the working chamber to maintain a stable CO2 concentration value. A sampling and monitoring port is installed on the back of the CO2 tank to facilitate the monitoring and sampling of concentration. Due to the adoption of a fast and slow dual inflation method, the CO2 concentration in the working chamber can be quickly recovered without overshooting after the CO2 tank is opened and closed.

According to the sensing methods of the CO2 gas path, there are two measurement methods: infrared sensor (IR) and thermal conductivity sensor (TC). Both sensors have advantages and disadvantages. The working principle of the thermal conductivity sensor for monitoring CO2 concentration is based on continuous measurement of the thermal conductivity of the inner chamber air. The low thermal conductivity of the input CO2 gas will cause changes in the thermal conductivity of the internal chamber air, which will generate an electrical signal directly proportional to the CO2 concentration. A disadvantage of the TC control system is that temperature and relative humidity changes within the box can affect the sensor's accuracy. When the door is frequently opened, not only does the CO2 concentration, temperature, and relative humidity also fluctuate considerably, concerning the accuracy of the TC sensor. This control system is unsuitable when precise cultivation conditions and frequent opening of incubator doors are required. Such a method is slowly eliminated in practice. Our new incubator generally uses the IR method for CO2 measurement and control. In actual practice, the TC method will waste more CO2, and it is a waste of material though TC seems cheaper a first glance.

Infrared sensor (IR) It detects CO2 levels through an optical sensor. The IR system includes an infrared emitter and a detector. When the CO2 in the tank absorbs some of the infrared rays emitted by the emitter, the sensor can detect the reduction in infrared rays. The amount of infrared rays absorbed corresponds to the level of CO2 in the tank, thereby obtaining the concentration of CO2 in the tank. Since the IR system determines the CO2 concentration in the tank through infrared reduction, and the particles in the tank can reflect or partially absorb infrared rays, making the IR system more sensitive to the number of particles in the tank, the IR sensor is more suitable for use in incubators with HEPA high-efficiency air filters at the air inlet.

Additional chemical adjustment methods for the pH value of carbon dioxide culture:

NaHCO3 tends to release CO2, and adding CO2 can inhibit this reaction. During cell culture, as the amount of CO2 released increases, the culture medium becomes acidic. Therefore, NaHCO3 (which forms a buffer pair with H2CO3 formed after CO2 is dissolved in water) is often added to adjust the pH.

The concentration of CO2 in the incubator should be balanced with the concentration of NaHCO3 in the culture medium. If the concentration of CO2 in the incubator is set at 5%, the amount of NaHCO3 added to the culture medium should be 1.97 g/L; If the CO2 concentration is maintained at 10%, the amount of NaHCO3 added should be 3.95 g/L.

Therefore, using carbon dioxide at a concentration of 5% is mainly to maintain the pH of the two-cell culture. (The above is a practical reference value, and the experimental personnel must adjust and record the particular situation based on the actual practical situation.) Of course, many specific requirements need to think for cell culture.

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