Somatic Cell Culture

Cell culture refers to a method of simulating the internal environment in vitro (sterility, suitable temperature, pH and certain nutritional conditions, etc.) to make it survive, grow, reproduce and maintain the main structure and function, cell culture is also called cell cloning technology. Cell culture is a key tool used in cell and molecular biology to model the physiology and biochemistry of cells^[1][2].

The cell culture proliferation process is divided into four phases: lag phase,log phase, plateau phase, and death phase. After the cells enter the new growth environment after inoculation, it takes a period of time to adapt to the environment, and the cells grow slowly during the lag phase.Due to the rich nutrition in the medium and less harmful substances discharged during the cell growth process, the cells quickly entered the log phase from the lag phase, and the cell growth showed exponential growth, showing a state of rapid growth. After a period of rapid growth and reproduction, the consumption of nutrients in the medium is increasing, harmful substances accumulate gradually, the number of cells has occupied the available matrix, there is no space for expansion, the cell proliferation rate will be greatly reduced or even completely stopped, and the cell will enter the plateau phase. In order to prevent cells from entering the death stage, the cells need to continue to grow and stimulate further proliferation.

Lag phase: Cells recover from sub-cultivation, attach to the surface and start to spread.

Log phase: Cells grow exponentially and double at a characteristic rate defining the cell line’s doubling time.

Plateau phase: The culture is confluent and cell growth slows or even stops.

Death phase: Cells start dying and detach from the surface.

MCE has not independently verified the accuracy of these methods. They are for reference only.

Different cells have different culture requirements. In order to achieve the best growth effect, it is necessary to simulate the in vivo environment in vitro and create a suitable environment for cell culture, which can be met by means of external conditions.

• Suitable cell culture medium

The medium is the most important component of the culture environment because it provides the necessary nutrients, growth factors and hormones for cell growth and regulates the pH and osmotic pressure of the culture^[4].

• Premium serum

Serum is an important nutrient in the process of cell culture, but also plays a certain role in cell protection. Serum is derived from animals and contains a variety of nutrients (plasma proteins, peptides, fats, carbohydrates, growth factors, hormones, inorganic substances, etc.) required for cell culture. Serum regulates the permeability of cell membranes, provides contact promoting and growth factors to protect cells from mechanical damage, and provides some protection to cells in culture. There are many types of serum (e.g. pig, rabbit, horse, sheep, etc.), but fetal bovine serum (FBS) is still the most widely used serum. FBS can provide rich growth factors that promote cell development and proliferation in vitro, and is the most widely used medium supplement for mammalian cell culture^[5][6].

• Constant cell growth temperature

To maintain the vigorous growth of cultured cells, there must be a constant and suitable temperature. Different types of cells have different requirements for culture temperature, and the optimal temperature for cell culture mainly depends on the body temperature of the cell host from which the cell is isolated^[7].

• Suitable gas environment

The culture of cells in vitro requires an ideal gas environment. Oxygen and carbon dioxide are necessary conditions for cell survival. Oxygen participates in the tricarboxylic acid cycle of cells, providing energy for cell survival, metabolism and synthesis; carbon dioxide is not only a metabolite of cells, but also an essential component for cell growth, and it is also related to maintaining the pH of the culture medium. CO₂ at 4-10% concentration is suitable for most cell culture experiments.

Most cells are anchorage-dependent and must be cultured attached to a solid or semisolid substrate (anchorage or monolayer culture), while others can grow floating in the medium (suspension culture).

• Subculture of Adherent Cells

1) Take out a bottle or a bottle of fine cells with good growth (the cell density reaches 70-90% is the best);

2) Use a sterile pipette to suck out the culture medium above the cells;

3) Wash the cells with a balanced salt solution that does not contain calcium and magnesium, tilt the culture flask or dish, and slowly add the balanced salt solution to cover the cells, draw a cross to wash, aspirate the waste liquid, and repeat the wash 2-3 times;

4) Add cell dissociation reagent to cover the cells with 0.5 mL,place the flask or dish in the cell culture incubator, the actual incubation time will vary with the cell line used. It is very important to control the time of the cell dissociation reagent. If the time is too short, the cells are still in an adherent state. If the time is too long, the protein on the cell surface will be damaged;

5) Place the culture bottle under a microscope for observation, and it is found that the cytoplasm retracts and the intercellular space increases, and the digestion is terminated;

6) Gently tilt the culture bottle or culture dish, the cell dissociation reagent will be aspirated, and the culture medium will be added. If only trypsin is used, a little serum-containing culture solution can be directly added to stop the digestion;

7) Use a sterile pipette to suck up the culture medium, blow and beat the cells on the bottle wall repeatedly to make the cells detach from the bottle wall and form a cell suspension, blow gently and not too hard;

8) Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 800-1000 rpm for 5 min;

9) Discard the supernatant, add new complete culture medium to the centrifuge tube, and pipette to form a cell suspension;

10) Count with a cell counter and inoculate in new culture flasks or culture dishes respectively.

• Suspension cell passages

The subculture process of suspension cells is simpler and easier to operate than adherent cells. Suspension cells are grown in culture medium without the need to dissociate from the container surface using cell dissociation reagents, the whole process is faster and less damaging to cells. For suspension cells, the medium can be changed directly, or the cells in the culture flask can be directly diluted and allowed to continue to expand.

1) Take out a bottle or a dish of well-growing cells, transfer the cell suspension to a 15 mL centrifuge tube, and centrifuge at 800-1000 rpm for 5 min;

2) Discard the supernatant, add new complete culture medium to the centrifuge tube, and pipette to form a cell suspension;

3) Count with a cell counter and inoculate in new culture flasks or culture dishes respectively.

1. Trypsin should be pre-warmed, preferably around 37℃.

2. The centrifugation speed should be appropriate. If the speed is too low, the cells cannot be separated effectively. If the centrifugation speed is too high and the time is too long, the cells will be squeezed, causing damage or even death.

3. Cells should be observed regularly, and if contamination is found, it should be dealt with in time.