Environmental Factors
-
Sterile Environment
- Being non-toxic and sterile is a primary requirement for in vitro cell culture. While cells in the living body have detoxification and immune systems to resist microbial or other harmful substance invasion, cells lose their defense capability against microorganisms and detoxification when cultured outside the body. To ensure cell growth and reproduction in an ex vivo environment, maintaining a sterile work area, good personal hygiene, sterile reagents and culture media, and aseptic procedures are crucial.Common microbial contaminants include mycoplasma, bacteria, and fungi. Mycoplasma, although non-lethal, can coexist with cells for a long time, potentially affecting them. Bacteria proliferate rapidly and can produce toxins that kill cells. Fungi, visible to the naked eye, can take various forms such as filamentous, tubular, or branching, floating on the surface of the culture medium.
-
Appropriate Temperature
- The suitable temperature for in vitro culture of mammalian and avian cells is generally 37-38°C. Inappropriate temperature can affect cell growth. Cells exhibit better tolerance to low temperatures than high temperatures; under low temperatures, metabolic activity and nuclear division capacity decrease. While temperatures not lower than 0°C may affect cell metabolism, placing cells at 40°C for several hours can be detrimental, even leading to cell death.
-
Suitable Osmotic Pressure
- High or low osmotic solutions can cause cells to wrinkle, swell, or rupture. Therefore, osmotic pressure is one of the important conditions for in vitro cell culture. Most cells in ex vivo culture have a certain tolerance to osmotic pressure, and in practical applications, an osmotic pressure of 260-320 mmol/L is suitable for most cells.
-
Gas Environment and pH
- Ideal gas conditions, including oxygen and carbon dioxide, are necessary for in vitro cell survival. Oxygen participates in the tricarboxylic acid cycle, providing energy for cell survival, metabolism, and synthesis. Carbon dioxide is both a metabolic byproduct and a necessary component for cell growth, also influencing the pH of the culture medium. The preferred pH range for most cells is often 7.2-7.4. In open cultivation, a 5% carbon dioxide gas ratio is suitable.
Nutritional Conditions
-
Culture Medium
- Culture media contain various nutrients required for cell growth, including carbohydrates, amino acids, inorganic salts, and vitamins. There are various synthetic culture media available, such as EBSS, Eagle, MEM, RPMI 1640, DMEM, tailored to the nutritional needs of different cells.
-
Other Additives
- In addition to providing basic nutritional substances in various synthetic culture media, other components need to be added based on different cell types and research purposes, such as serum, growth factors, etc.Serum provides extracellular matrix, growth factors, transferrin, and other important substances. Fetal bovine serum is commonly used, and the proportion of serum added should be determined based on the specific cell type and research goals. A 10%-20% serum concentration supports faster cell growth and is termed growth medium, while adding 2%-5% serum maintains slow growth or non-senescence, known as maintenance medium. However, serum contains substances harmful to cell growth and reproduction, such as complement, immunoglobulins, and some growth inhibitors. Its unclear composition may impact result analysis, and variations in serum activity between different animals and batches can affect the stability of culture results.Glutamine is an essential nitrogen source for cell growth, playing a crucial role in cellular metabolism. However, due to its instability and susceptibility to degradation in solution, glutamine needs to be added before use.To prevent contamination, a certain amount of antibiotics should be added to the culture medium. Commonly used antibiotic names, concentrations, and sensitivities are provided in the table below.
