The quality of culture medium is the key to the development of serum cell culture, and animal serum, the main component of culture medium, plays an important or even irreplaceable role in cell growth and reproduction. Bovine serum is the most widely used animal serum, so serum is one of the important raw materials for pharmaceutical biotechnology products. Ensuring serum quality is also an important link to improve the quality of biological products. 1. Serum type
At present, the serum used for tissue culture is mainly bovine serum, and some special cells are also cultured with human serum and horse serum. The reasons for choosing bovine serum cultured cells are: sufficient sources, mature preparation technology, and people have a deeper understanding of it after long-term application tests. Bovine serum is suitable for most mammalian cells, but it is not excluded that it is more suitable to use other animal serum when cultivating some cells.
Bovine serum is the most widely used natural medium in cell culture, which contains rich nutrients necessary for cell growth and has extremely important functions. Bovine serum is divided into calf serum, new bovine serum and fetal bovine serum. Fetal calf serum should be taken from the fetal calf delivered by caesarean section; The serum of newborn cows was taken from newborn cows within 24 hours after birth; Calf serum was obtained from calves born at 10-30 days. Obviously, the quality of fetal calf serum is the highest, because the fetal calf has not been in contact with the outside world, and the serum contains the least harmful components to cells such as antibodies and complements. 2. The main components of serum
Serum is a very complex mixture, which is formed by removing fibrin from plasma. Although most of its components are known, some of them are still unknown. The composition and content of serum often vary with the gender, age, physiological status and nutritional status of blood donors. Serum contains various plasma proteins, peptides, fats, carbohydrates, growth factors, hormones, inorganic substances and so on. They are in physiological balance in promoting cell growth or inhibiting growth activity. Although great progress has been made in the study of serum composition and function, there are still some problems. The main points are as follows: First, there may be hundreds of serum components, but the exact composition, content and mechanism of action are still unclear, especially some polypeptide growth factors, hormones and lipids are not fully understood, which brings many difficulties to the research work. Secondly, serum is produced in batches, which varies greatly from batch to batch, and the storage period of serum is at most one year. Therefore, it is extremely difficult to ensure the similarity of each batch of serum, which limits the standardization and continuity of the experiment. Thirdly, it is not excluded that serum contains volatile substances, which is considered to be one of the reasons for "deterioration in the bottle". 3. The main role of serum
Provide basic nutrients: amino acids, vitamins, inorganic substances, lipid substances, nucleic acid derivatives, etc. , is an essential substance for cell growth.
Provide hormones and various growth factors: insulin, adrenocortical hormones (hydrocortisone, dexamethasone), steroid hormones (estradiol, testosterone, progesterone), etc. Growth factors, such as fibroblast growth factor, epidermal growth factor and platelet growth factor.
Provide binding proteins: binding proteins are used to carry important low molecular weight substances, such as albumin, vitamins, fats and hormones, and transferrin carries iron. Binding proteins play an important role in cell metabolism.
Provide contact promotion and stretching factors to prevent cell adhesion from being mechanically damaged.
It has a certain protective effect on cells in culture: some cells, such as endothelial cells and bone marrow-like cells, can release protease, and serum contains anti-protease components, which play a neutralizing role. This effect was discovered by accident, and now serum is purposefully used to stop the digestion of trypsin. Because trypsin has been widely used in the digestion and passage of adherent cells. Serum protein forms the viscosity of serum, which can protect cells from mechanical damage, especially in suspension culture and stirring, and viscosity plays an important role. Serum also contains some trace elements and ions, which play an important role in metabolism and detoxification, such as SeO3.
Selenium and so on. 4. Disadvantages of using serum in cell culture
The composition of serum is very complicated. Although it contains many components beneficial to cells, it also contains components harmful to cells, which makes serum have several obvious shortcomings:
For most cells, in the body, serum is not the physiological liquid they contact, but in the process of injury healing and blood coagulation, so the use of serum may change the normal state of some cells in the body, and serum may promote the growth of some cells (fibroblasts) and inhibit the growth of other cells (epidermal cells).
Serum contains some substances that are toxic to cells, such as polyamine oxidase, which can react with polyamines (such as spermine and spermidine) produced by highly proliferative cells to produce spermidine with cytotoxic effect. Complement, antibody and bacterial toxin will all affect cell growth and even lead to cell death.
Different animals have different serum sources and batch numbers, and the quality of each batch is very different, so its composition cannot be consistent.
Mycoplasma and virus may be brought into the sample, which may have a potential impact on cells and may lead to the failure of the experiment or the unreliable experimental results.
The use of serum makes it difficult to standardize experiments and production, and protein makes it difficult to separate and purify some transgenic protein biopharmaceuticals.
In mass production, the source of serum is becoming more and more difficult and expensive, which is one of the main parts of the production cost of animal cell culture.