Cell culture technology is also called cell cloning technology, and the formal term in biology is cell culture technology. Cell culture is an indispensable process for the whole bioengineering technology or one of the biological cloning technologies, and cell culture itself is a large-scale cloning of cells. Cell culture, including microbial cell culture, also includes? To the oval office? Shore? Participate? Nap? Cell culture technology can transform a cell into a simple single cell or multi-cell with little differentiation after a large number of cultures, which is an essential link of cloning technology, and cell culture itself is cell cloning. A large number of cells or their metabolites are obtained by cell culture. Because biological products are all derived from cells, it can be said that cell culture technology is the most core and basic technology in biotechnology.
The growth of cells needs a certain nutritional environment, and the nutrient matrix used to maintain cell growth is called culture medium. According to the physical state, media can be divided into liquid media and solid media. Liquid culture medium is used in large-scale industrial production and research on basic theories such as physiological metabolism. Liquid culture medium is made by adding some coagulant (such as agar) or solid culture (such as bran and rice). Solid culture medium provides a nutritious and ventilated surface for cell growth, and the cells produced on this nutritious surface can form a single colony. Therefore, solid culture medium plays a very important role in cell separation, identification and counting. In order to isolate the needed cells from multicellular organisms and transform them in vitro, the problem of cell culture in vitro should be solved first. Compared with the difficulty of microbial cell culture, it is more difficult to culture single cells from multicellular organisms, especially animal cells.
1, general conditions of cell culture
In short, cells can provide anything they need. It takes time to really do this. So far, people do not know enough about the life cycle control mechanism of cells. Although cancer cells also come from normal cells, they still don't know why it is difficult for cancer cells to stop the harmful division that has begun. Nevertheless, people's long-term research results show that the basic conditions for cell culture in vitro are the following cell physiological conditions.
(1) temperature
If the temperature is too low, the cells will grow slowly or even not. Cryopreservation of cells can maintain the original ability of cell division and differentiation. Excessive temperature will lead to cell death. This is mainly determined by the optimum temperature required by enzyme and protein. Most biological macromolecules are easy to change or lose their spatial structure (denaturation) after encountering high temperature. Cell membrane is easily deformed at high temperature. In nature, there are both high-temperature resistant cells and low-temperature resistant cells. It is of great significance to study the mechanism of biological growth under extreme conditions to cope with extreme environment in biological evolution, agriculture, environmental protection and fermentation industry.
(2) pH value
Excessive acid or alkali can lead to cell death. This is mainly related to the degeneration of protein and the structural damage of cell membrane.
(3) Osmotic pressure
The proportion and types of water-soluble substances inside and outside the cell determine the degree of expansion and contraction of the cell, because the cell membrane is a semi-permeable membrane, and only substances beneficial to it are allowed to pass through. The distribution of the same substance inside and outside the cell is different. When the concentration of a water-soluble substance is too high outside the cell, it may lead to cell withering and death, while when the concentration of these substances is too high inside the cell, it may lead to excessive water absorption and expansion of the cell. The ability of cell membrane to regulate osmotic pressure is limited.
(4) nutrients
Nutrients and water are also called cell culture solution, which contains various substances needed for cell proliferation and growth. Nutrients include: N source and C source, which are related to providing energy; Inorganic salts, vitamins and hormones are related to metabolic regulation. The design of cell culture medium has always been the key of cell culture technology in vitro. The ideal cell culture solution can meet all the needs of cell culture in vitro for pH, osmotic pressure, nutrients and regulatory substances. In the research and application of stem cell differentiation, the key is to find a nutrient solution to differentiate stem cells into needed cells and tissues. The same human stem cells were put into different nutrient solutions to differentiate and cultivate various human organs, and this past dream began to come true. Plant cell tissue culture technology has been basically improved. With the continuous improvement of plant cell and tissue culture technology, such as precious flowers, Chinese herbal medicines, virus-free potatoes, tissue culture lotus seedlings, etc., especially because of the commercialization of tissue culture solution, it has been widely accepted by farmers.
(5) Water
Water is the largest substance needed by cells, and the water content of cells varies greatly with different species, different parts and different growth periods. The water content of dried plant cells is as high as 90%. The demand for water is usually considered together with the cell culture solution.
(6) Aseptic conditions
Cell culture in vitro is only the cells needed for culture, and there are various other microorganisms in the environment (such as air), so the cells needed must be isolated and cultured without mixed bacteria. Aseptic condition is the most basic condition for cell culture in vitro.
(7) light
Plant cells and some bacteria need light for photosynthesis.
(8) Natural gas
Animal cells need continuous oxygen supply and carbon dioxide elimination, while plant cells do the opposite.
2. Special conditions for animal cell culture
Among all in vitro cell cultures, animal cell cultures are the most difficult. The following are the special conditions it needs.
(1) Serum: Serum is often needed for animal cell culture in vitro. The most commonly used is calf serum. Serum provides essential factors for growth, such as hormones, trace elements, minerals and fats. One day people really learned to prepare the same culture solution as serum, and then serum can replace it. Here, serum is equal to the natural nutrient solution of animal cells cultured in vitro.
(2) Support: Most animal cells have the habit of adhering to the wall. In vitro culture usually uses glass and plastic as supports.
(3) Gas exchange: In the process of cell culture, the ratio of carbon dioxide and oxygen should be constantly adjusted to maintain the required gas conditions. How difficult is it to quickly restore the equipment after each unpacking operation? Therefore, animal cell culture equipment in vitro requires high requirements and large investment.
3. Special conditions for plant cell culture
(1) illumination: The light conditions of plant cells cultured in vitro are not strict, because the substances needed for cell growth are mainly supplied by the culture medium. However, light is not only related to photosynthesis, but also to cell differentiation. For example, photoperiod can regulate sexual cell differentiation and flowering, so light conditions are particularly important in the early plant cell culture process for the purpose of obtaining plants. The process of obtaining important substances (such as drugs) by in vitro culture of plant cells, most of which are suspended in a reactor.
(2) Hormones: The division and growth of plant cells need the regulation of plant hormones, and auxin and mitogen which promote growth are the most basic hormones. The division, growth, differentiation and individual growth cycle of plant cells are regulated by corresponding hormones. Compared with animal cells, the principle of hormone demand of plant cells in vitro culture has been understood, its application technology has been quite mature, and a set of culture medium has been widely used as a commodity. At the same time, it solves the demand of plant cells for water, nutrition, hormones, osmotic pressure, pH and trace elements.
4. Special conditions for microbial cell culture
Microorganisms are mostly single-celled organisms, and the living conditions in the field are relatively simple. Therefore, the conditions for artificial cultivation of microorganisms are much simpler than those of animal and plant cells. Among them, the cultivation of anaerobic microorganisms is more complicated than aerobic microorganisms, because strict anaerobic needs to maintain the concentration of non-oxygen inert gases such as carbon dioxide, while aerobic microorganisms only need to provide sterile oxygen through continuous stirring. The requirements of microorganisms on culture conditions are not as good as those of animal and plant cells, and corn steep liquor, peptone, wort and yeast extract become good natural culture media for microorganisms. For the nutritional requirements of some special microorganisms, they can be added on the basis of these natural media.