cell culture refers to a method of simulating the in vivo environment (aseptic, suitable temperature, pH level and certain nutritional conditions, etc.) in vitro to enable it to survive, grow, reproduce and maintain its main structure and function. Cell culture, also known as cell cloning technology, is formally referred to as cell culture technique in biology. Whether for the entire biotechnology industry or one of its technologies, biocloning, cell culture is an essential process. Cell culture itself is the large-scale cloning of cells. Cell culture technology can transform a single cell into a simple single cell or a multi-cell with very little differentiation through large-scale cultivation. This is a crucial step in cloning technology, and cell culture itself is the cloning of cells. Cell culture technology is an important and commonly used technique in cell biology research methods. Through cell culture, not only can a large number of cells be obtained, but also the signal transduction, anabolism, growth and proliferation of cells can be studied.

Now I'll tell you all about the environmental factors and nutritional conditions involved in cell culture!

Environmental factors:

1. Sterile environment

Non-toxicity and sterility are the primary conditions for cell culture in vitro. In the living body, the detoxification system and immune system of cells can resist the invasion of microorganisms or other harmful substances. However, during the process of cell culture in vitro, cells lack the protection of the body's immune system and lose their defense ability against microorganisms and detoxification ability against harmful substances. To ensure that cells can grow and reproduce in an in vitro environment, it is necessary to guarantee a sterile working area, good personal hygiene, sterile reagents and culture media, as well as aseptic operation.

Common microbial contaminants include mycoplasma, bacteria and fungi. Mycoplasma has no lethal toxicity and can coexist with cells for a long time, having a potential impact on cells. However, it is small in size and difficult to identify. It can be detected by lichen red or Hoechst33342 staining. Bacteria multiply rapidly and can expand in large quantities within a short period of time, and produce toxins to kill cells. There are numerous types of fungi, which are visible to the naked eye and float on the surface of the culture medium. They can take on filamentous, tubular or dendritic forms, etc.

2. Appropriate temperature

The suitable temperature for the in vitro culture of general mammalian and avian cells is 37 to 38 degrees Celsius. Unsuitable environmental temperatures can affect the growth of cells. Cells have a stronger tolerance to low temperatures than to high temperatures. At low temperatures, the metabolic activity and mitotic ability of cells decrease. If the temperature is not lower than 0℃, although the cell metabolism is affected, there is no damaging effect. At 25-35℃, cells grow at a slow rate. However, if they are placed at 40℃ for several hours, it is not only detrimental to the survival and growth of the cells, but may even lead to their death.

3. Appropriate osmotic pressure

Hypertonic or hypotonic solutions can cause cells to wrinkle, swell and rupture. Therefore, osmotic pressure is one of the important conditions for cell culture in vitro. Most cells cultured in vitro have a certain tolerance to osmotic pressure. In practical applications, an osmotic pressure of 260 to 320mmol/L can be applied to the majority of cells.

4. Gas environment and pH

The in vitro culture of cells requires an ideal gas environment. Oxygen and carbon dioxide are necessary conditions for the survival of cells. Oxygen participates in the tricarboxylic acid cycle of cells, providing energy for cell survival, metabolism and synthesis. Carbon dioxide is not only a metabolic product of cells and an essential component for cell growth, but also related to maintaining the pH of the culture medium. The suitable pH range for most cells is often 7.2 to 7.4. In open culture, a proportion of 5% carbon dioxide gas is appropriate. [1]

Nutritional conditions:

1. Culture medium

Cell culture medium contains various nutrients required for cell growth, including carbohydrates, amino acids, inorganic salts, vitamins, etc. There are various synthetic culture media available to meet the nutritional requirements of different cells, such as EBSS, Eagle, MEM, RPMll640, DMEM, etc.

2. Other added ingredients

In addition to providing basic nutrients through various synthetic culture media, other components such as serum and factors also need to be added according to different cells and different culture purposes. Serum provides important substances such as extracellular matrix, growth factors and transferrin. The commonly used one is fetal bovine serum. The proportion of serum to be added should be determined based on different cells and different research purposes. 10% to 20% of serum can maintain a relatively fast growth and proliferation rate of cells and is called growth culture medium. To maintain the slow growth or immortality of cells, adding 2% to 5% of serum is sufficient, which is called a maintenance culture medium. However, there are also substances in the serum that are harmful to cell growth and reproduction, such as complement, immunoglobulin and some growth inhibitory factors; The components are not clear, which affects the analysis of the results. The serum activities of different animals and different batches vary greatly, affecting the stability of the culture effect. Glutamine is an important nitrogen source for cell growth and plays a significant role in the metabolic process of cell growth. However, as glutamine is very unstable and prone to degradation in solution, it can decompose about 50% after being placed at 4℃ for 7 days. Therefore, glutamine needs to be added before use.