The Impact of pH on Cell Culture Media

News 31 7 月, 2024

Cell culture media are vital for providing the necessary nutrients and environment for cell growth and proliferation. The pH of the media is a crucial factor that can significantly affect cell health and experimental outcomes. While cell culture techniques are generally similar, the specific conditions required for different cell types can vary widely. Deviation from the optimal conditions can result in altered cell phenotypes.

Key Components and Functions of Cell Culture Media

1. Glucose: Serves as the primary energy source for cell metabolism.

2. CO₂: Cells require a small amount of carbon dioxide to maintain their growth.

In the culture media, dissolved CO₂ is in equilibrium with bicarbonate ions. Many media use the CO₂/bicarbonate buffer system to maintain the pH. CO₂ dissolves in the media and reacts with water to form carbonic acid. As cell metabolism produces more CO₂, the reaction shifts to the right, lowering the pH of the medium.

By adding sodium bicarbonate to the culture media, the pH can be maintained within the optimal range of 7.2 to 7.4.

3. Phenol Red: Used as a pH indicator in the media.

As cells grow, their metabolic by-products change the pH, which in turn changes the color of the medium. At lower pH values, phenol red turns yellow, and at higher pH values, it turns purple. For most culture systems (pH 7.2 to 7.4), the medium should be bright red. Some special media do not include phenol red, especially for estrogen-sensitive breast tissue cells, as phenol red can mimic steroid functions and affect cells.

The Importance of pH in Cell Culture

Maintaining a stable pH is crucial for cell culture. The optimal pH range is generally between 7.2 and 7.4. Media that are too acidic or too alkaline can adversely affect cell health.

Most cell lines grow best at a pH around 7.4. Phenol red, the common pH indicator in media, gives the medium its characteristic red color. Different media have varying colors; for example, DMEM is darker than 1640. The color of the medium can also indicate cell health.

• Yellow Medium: Indicates acidity, which could be due to:
  • Overgrowth of cells
  • Bacterial contamination
  • High CO₂ concentration in the incubator
• Purple Medium: Indicates alkalinity, which could be due to:
  • Slow or no cell growth
  • Fungal contamination
  • Low CO₂ concentration in the incubator
    
    

Additional Key Components in Cell Culture Media

1. L-Glutamine: An essential amino acid for most mammalian and insect cells, necessary for protein synthesis and nucleic acid metabolism. It is unstable and degrades over time, depending on storage conditions.

2. Non-Essential Amino Acids (NEAA): Including amino acids like alanine, asparagine, aspartic acid, glycine, glutamic acid, proline, and serine, NEAAs reduce metabolic burdens and enhance cell proliferation.

3. Sodium Pyruvate: An intermediate in glycolysis, it can be used as an energy source and carbon skeleton for metabolic processes. Most media contain 1 mM sodium pyruvate, but in L-15 medium, the concentration is increased to 5 mM for use in CO₂-free environments.

Common Additives in Cell Culture Media

1. Penicillin-Streptomycin (Pen/Strep): Prevents bacterial contamination.

2. Insulin: Promotes cell proliferation, extends cell lifespan, and increases protein and nucleic acid expression.

3. Insulin-Transferrin-Selenium (ITS): Reduces the need for fetal bovine serum in cell maintenance and low-density adherent cultures.

4. Dexamethasone: An anti-inflammatory glucocorticoid that influences cell survival, signaling pathways, and gene expression.

5. Hydrocortisone: Enhances proliferation of epidermal and mammary epithelial cells and improves colony formation of neural glial and fibroblast cells.

6. Interleukin-2 (IL-2): A T-cell growth factor that enhances NK cell cytolytic activity and promotes B-cell immunoglobulin production, commonly used in NK92 cell cultures.

Common Issues in Cell Culture

1. Ineffective Culture Media: Regularly check the expiration date and ensure the correct media are used for specific cell types (e.g., high-glucose DMEM for 293T cells and RPMI 1640 for THP-1 cells).
2. Storage: Store media at 4°C and aliquot to avoid repeated temperature changes, which accelerate glutamine degradation.
3. Color Variations: Different media have distinct colors (e.g., DMEM is redder than MEM). Observe color changes to assess cell health.
4. Media Acidification: If the medium turns orange or yellow, replace it promptly as it indicates significant acid production from cell metabolism.
5. Gas Exchange Effects: When media are exposed to air during opening or when removing culture plates/bottles from the incubator, the color may change to purple-red. This can be corrected by returning the media to the CO₂ incubator to restore normal pH and color.
   
   

Conclusion

Maintaining the correct pH in cell culture media is essential for optimal cell growth and function. Understanding the role of various components and additives helps ensure successful cell culture experiments, leading to reliable and reproducible results in biological and medical research.