(1) Nutritional requirements

·Amino acids -12 essential amino acids (valine, leucine, isoleucine, threonine, lysine, tryptophan, phenylalanine, methionine, histidine, tyrosine, arginine, cysteine); 10 non essential amino acids; In addition, glutamine is also required, which plays an important role in cellular metabolism. The nitrogen it contains is the source of purine and pyrimidine synthesis in nucleic acids, and is also the basic substance required for the synthesis of triphosphate, diphosphate, and monophosphate glycosides.

·Carbohydrates - Carbohydrates - Glucose; Galactose; Cells have the highest absorption capacity for glucose and low for galactose.

·Inorganic salts - sodium, potassium, magnesium, phosphorus, calcium, etc;

·Vitamins - Biotin, Folic Acid, Riboflavin, Vitamin C, Vitamin B12, Pyridoxine, etc;

·Proteins and peptides - albumin, aprotinin, fetoprotein, fibronectin, transferrin, growth factors;

·Fatty acids and lipid substances - linoleic acid, oleic acid, cholesterol, etc;

·Trace elements - iron, zinc, selenium, copper, manganese, molybdenum, vanadium, etc.

(2) Temperature

The commonly used temperature is 37 ° C, but it should be noted that the most suitable culture temperature varies for tissue cells from different biological sources, and cells are tolerant to low temperatures but not high temperatures. Within the physiological temperature range, temperature is positively correlated with cell growth rate.

(3) Osmotic pressure

The magnitude of osmotic pressure is mainly determined by the concentration of various salt components in the culture medium. Cells must live in an environment that is isotonic with their own protoplasts. If the osmotic pressure of the living environment is higher than their own, cultured cells may face dehydration and die. If the osmotic pressure of the living environment is lower than their own, cultured cells may face water absorption and rupture and die. However, most cultured cells have a certain tolerance to osmotic pressure. The osmotic pressure of human plasma is 290mOsm/kg, which can be considered as the ideal osmotic pressure for cultivating human cells. The osmotic pressure of mouse cells is around 320mOsm/kg. For cultivating most mammalian cells, it is advisable to control the osmotic pressure of the culture medium within the range of 260-320 mOsm/kg.

(4) Gas phase

The gas phase environment for in vitro cell culture is often composed of 5% CO2 and 95% air. O2 is involved in the energy metabolism process of cells. CO2 is used to maintain the pH of the culture medium.

(5) pH

The pH of the culture medium suitable for the growth of most cells is between 7.0-7.4, and an environment with pH 7.4 is suitable for most mammalian cell lines. Phenol red is used as a pH indicator in culture media to continuously monitor the acidity and alkalinity of the culture medium. At low pH values, phenol red makes the culture medium yellow, while at higher pH values, it makes the culture medium purple. At pH values of 7.2-7.4, it is red and suitable for cell culture.

(6) Non toxic and pollution-free

Non toxicity and sterility are prerequisites for in vitro cell culture. In the detoxification and immune systems of living cells, they can resist the invasion of microorganisms or other harmful substances. However, in vitro culture, cells lack the protection of the body's immune system, resulting in the loss of defense against microorganisms and detoxification ability against harmful substances. Therefore, ensuring a non-toxic and pollution-free environment for cell growth is the primary condition for successful cell culture. During the cell culture process, it is important to prevent contamination by toxic chemicals, microorganisms, and cross contamination between different cells.