FAQs of Cell Culture
- What is cell culture?
Cell culture is a process in which cells are grown under controlled conditions. Cells can be microorganisms such as bacteria and yeast, or human, plant or animal cells. - What are the advantages of cell culture?
The physicochemical environment of cell culture (i.e. pH, temperature, osmotic pressure, oxygen and carbon dioxide tension) is highly controllable and the control of physiological conditions can be constantly checked. - What are the steps of cell culture?
- Thawing
- Cell Seeding
- Cell Observation
- Cell passaging
- Stock Preparation
- What are the Basic Constituents of Media?
- Inorganic salts
- Carbohydrates
- Amino acids
- Vitamins
- Fatty acids and lipids
- Proteins and peptides
- Serum
- Trace elements
- Why does the pH of the culture solution change rapidly?
- Why does the culture solution appear to precipitate?
- Why do cultured cells die and grow abnormally?
- Lack of CO2 in the incubator, or too much temperature fluctuation in the incubator.
- Cells are damaged during freezing or resuscitation, and new preserved cell species need to be taken.
- Incorrect culture medium osmolarity. It should be noted that most mammalian cells can tolerate an osmolarity of 260 - 350 mOsm/kg. Adding additional reagents such as HEPES or drugs may affect the culture medium osmolarity.
- Accumulation of toxic metabolites in the culture medium requires prompt replacement with fresh culture medium.
- Why is the growth of cultured cells slowed down?
- Cell density is too low during passaging, and the inoculation density of cells can be increased.
- If the cell state is aging, replace the cells with new ones in time.
- If mycoplasma contamination is suspected, be sure to isolate the culture dish suspected of contamination and clean and disinfect the incubator in a timely manner.
- It may be that certain growth factors are missing from the culture medium because of the specific type of cells.
- Why serum is used in cell culture?
Serum is vitally important as a source of growth and adhesion factors, hormones, lipids, and minerals for the culture of cells in basal media. - What is the best way to store the serum?
Serum should be stored at -5℃ to -20℃. If stored at 4°C, do not store it for more than one month. If only a portion of the serum is taken at a time, it is recommended to aseptically dispense the serum into a proper sterilized container and then put it back into the freezer. - How do I thaw the serum?
It is recommended that the serum be removed from the freezer and placed in a refrigerator at 2 to 8°C to thaw, and then at room temperature to thaw completely. Note that the serum should be shaken evenly during the thawing process. - Why do flocculent precipitates appear after serum thawing?
There are many reasons for the appearance of precipitates in serum, the most common reason being denaturation of lipoproteins in serum. In addition, fibrin is also present in serum after thawing and is one of the main causes of precipitates. However, these flocculent precipitates do not affect the quality of the serum itself. - How to perform cell detachment?
Trypsin is the most commonly used isolating agent. During this process, most surface proteins are destroyed and excessive trypsinization can lead to irreversible cell damage. Conversely, inadequate trypsinization leads to incomplete cell harvesting.
| Cause | Solution |
|---|---|
| Problem of CO2 tension. | Increase or decrease the CO2 concentration in the incubator according to the NaHCO3 concentration in the culture medium. 2.0 g/L to 3.7 g/L NaHCO3 concentration corresponds to a CO2 concentration of 5% to 0%. Alternatively, switch to a CO2-independent culture medium. |
| Problem of NaHCO3 buffer solution. | Add HEPES buffer to a final concentration of 10 to 25 mM. |
| Problem of salt concentration in the culture medium. | Incubate in a CO2 culture environment with a culture solution based on Earle's salt and in an atmospheric culture environment with a culture solution based on Hank's salt. |
| Bacterial, yeast or fungal contamination. | Sterilize with antibiotics, or discard the culture. |
| Appearance | Cause | Solution |
|---|---|---|
| The culture solution appears to precipitate while the pH remains unchanged | Residual phosphate after detergent cleaning precipitates the culture medium components. | Glassware is repeatedly rinsed with deionized water and then sterilized. |
| The culture solution appears to be precipitated and the pH value changes | Bacterial or fungal contamination. | Sterilize with antibiotics, or discard the culture. |
