One of the main advantages of cell culture is the ability to manipulate the physical chemistry of cell reproduction (ie temperature, pH, osmotic pressure, O2 and CO2 tension) and physiological environment (ie hormone and nutrient concentration). In addition to temperature, the culture environment is controlled by the growth medium.
Although the physiological environment of culture is not as clear as its physical and chemical environment, a better understanding of serum components, identification of growth factors required for proliferation, and a better understanding of the microenvironment of cells in culture. (I.e. cell-cell interaction, gas diffusion, interaction with the matrix) now allows certain cell lines to be cultured in serum-free media.
1.Culture environment affects cell growth
Please note that the cell culture conditions are different for each cell type.
The consequences of deviating from the culture conditions required for a particular cell type range from the expression of abnormal phenotypes to the complete failure of cell culture. Therefore, we recommend that you become familiar with the cell line you are interested in and strictly follow the instructions provided for each product you use in your experiment.
2.Precautions for creating an optimized cell culture environment for your cells:
Culture media and serum (see below for more information)
pH and CO2 levels (see below for more information)
Cultivate plastic (see below for more information)
Temperature (see below for more information)
2.1 Cultural Media and Serum
The culture medium is the most important part of the culture environment, because it provides the nutrients, growth factors and hormones needed for cell growth, and regulates the pH and osmotic pressure of the culture.
Although the initial cell culture experiments were performed using natural media obtained from tissue extracts and body fluids, the need for standardization, media quality, and increased demand led to the development of definitive media. The three basic types of media are basal media, reduced serum media and serum-free media, and they have different requirements for serum supplementation.
2.1.1 Basic medium
Gibco cell culture medium
Most cell lines grow well in basic media containing amino acids, vitamins, inorganic salts, and carbon sources (such as glucose), but these basic media formulations must be supplemented with serum.
2.1.2 Reduced serum medium
Bottle with Gibco Low Serum Medium
Another strategy to reduce the adverse effects of serum in cell culture experiments is to use serum-reduced media. Reduced serum medium is a basic medium formula rich in nutrients and animal-derived factors, which can reduce the amount of serum required.
2.1.3 Serum-free medium
Bottle with Gibco serum-free medium
Serum-free medium (SFM) circumvents the use of animal serum by replacing serum with appropriate nutrition and hormone formulations. Many primary cultures and cell lines have serum-free medium formulations, including the Chinese Hamster Ovary (CHO) recombinant protein production line, various hybridoma cell lines, insect lines Sf9 and Sf21 (Spodoptera frugiperda), as well as for The host for virus production (for example, 293, VERO, MDCK, MDBK), etc. One of the main advantages of using a serum-free medium is the ability to make the medium selective for specific cell types by selecting an appropriate combination of growth factors. The following table lists the advantages and disadvantages of serum-free media.
Advantage
Increase clarity
More consistent performance
Easier purification and downstream processing
Accurately assess cell function
Increase productivity
Better control of physiological reactions
Enhanced cell media detection
Disadvantage
Cell type specific medium formula requirements
Need higher reagent purity
Slowdown in growth
2.2.1 pH level
Most normal mammalian cell lines grow well at pH 7.4, and the differences between different cell lines are small. However, some transformed cell lines have been shown to grow better in a slightly acidic environment (pH 7.0 – 7.4), while some normal fibroblast cell lines prefer a slightly alkaline environment (pH 7.4 – 7.7). Insect cell lines such as Sf9 and Sf21 grow best at pH 6.2.
2.2.2 CO2 level
The growth medium controls the pH of the culture and buffers the cells in the culture to resist changes in pH. Usually, this buffering is achieved by containing organic (for example, HEPES) or CO2-bicarbonate-based buffers. Because the pH of the medium depends on the delicate balance of dissolved carbon dioxide (CO2) and bicarbonate (HCO3-), changes in atmospheric CO2 will change the pH of the medium. Therefore, when using a medium buffered with a CO2-bicarbonate-based buffer, it is necessary to use exogenous CO2, especially when culturing cells in open culture dishes or culturing transformed cell lines at high concentrations. Although most researchers usually use 5-7% CO2 in the air, most cell culture experiments usually use 4-10% CO2. However, each medium has a recommended CO2 tension and bicarbonate concentration to achieve the correct pH and osmotic pressure; for more information, please refer to the medium manufacturer’s instructions.
2.3 Cultivating plastics
Cell culture plastics are available in a variety of forms, sizes and surfaces to suit various cell culture applications. Use the cell culture plastic surface guide and cell culture container guide below to help you choose the right plastic for your cell culture application.
View all Thermo Scientific Nunc cell culture plastics (advertising link)
2.4 Temperature
The optimal temperature for cell culture depends to a large extent on the body temperature of the host from which the cells are isolated, and to a lesser extent on the anatomical changes in temperature (for example, skin temperature may be lower than that of skeletal muscle). For cell culture, Overheating is a more serious problem than overheating. Therefore, the temperature in the incubator is usually set slightly below the optimum temperature.
2.4.1 Optimum temperature for various cell lines
Most human and mammalian cell lines are kept at 36°C to 37°C for optimal growth.
Insect cells are cultivated at 27°C for optimal growth; they grow more slowly at lower temperatures and temperatures between 27°C and 30°C. Above 30°C, the vitality of insect cells decreases, even if it returns to 27°C, the cells will not recover.
Avian cell lines need 38.5°C to reach maximum growth. Although these cells can be kept at 37°C, they will grow more slowly.
Cell lines derived from cold-blooded animals (such as amphibians, cold-water fish) can tolerate a wide temperature range of 15°C to 26°C.
Post time: Feb-01-2023