Common Additives in Cell Culture Media: An Overview
News 31 7 月, 2024
Cell Culture Additives
Cell culture is an essential experimental method in biological research, providing the nutrients and environment necessary for cell growth and proliferation. Cell culture media are crucial in this process, serving as both the nutritional supply and living environment for cells.
Typically, serum is added to the cell culture medium to maintain cell growth and survival. However, in addition to serum, other components like insulin and β-mercaptoethanol are also required for certain cells to maintain optimal conditions. For example, cells like MCF-7 and NIHrequire additional insulin, while cells like Thp-1 and MC3T3-E1 subclone 14 need β-mercaptoethanol. The required amounts vary depending on the cell type.
Different media, with various additives, can be used for different cell cultures and experimental needs. Let’s delve into the functions of various additives commonly used in cell culture media.
Introduction to Cell Culture Additives
1. The Importance of L-Glutamine in Cell Culture
L-Glutamine is an essential amino acid necessary for cell growth, serving as an energy source and participating in protein synthesis and nucleic acid metabolism. However, L-Glutamine is unstable in solution, with its degradation rate affected by storage time, temperature, and pH. L-Alanyl-L-Glutamine is a stable alternative widely used in cell culture, providing consistent nutrition over time and reducing cell toxicity caused by degradation products. This stable solution is suitable for mammalian adherent or suspension cell cultures, offering better control over culture conditions and enhancing cell vitality.
2. Functions and Usage of Glutamine in Media
Almost all cells require high levels of glutamine for protein synthesis. Without sufficient glutamine, cells cannot grow well and may die. Therefore, most culture media contain a significant amount of glutamine. Due to its instability in solution, it should be stored at -20°C and added to the medium before use. If stored in liquid media at 4°C for over two weeks, fresh glutamine should be added.
3. What is GlutaMAX-I and Its Stability?
GlutaMAX-I, an L-glutamine derivative, protects the unstable alpha-amino group with L-alanine. It is gradually hydrolyzed by peptidases, releasing L-glutamine for use. GlutaMAX-I is highly stable, with minimal degradation even after sterilization at 121°C for 20 minutes, unlike L-glutamine, which nearly completely degrades under the same conditions.
4. The Role of Sodium Pyruvate in Culture Media
Cells primarily use glucose as a carbon source, but sodium pyruvate can serve as an alternative. When glucose is insufficient, cells metabolize sodium pyruvate instead. Pyruvate, an intermediate in glycolysis, provides energy and a carbon backbone for metabolism. Some cells require pyruvate for survival, proliferation, and growth in low-serum conditions. Typically, media contain 0.1 mM sodium pyruvate, with commercial preparations often being 10 mM (100X) solutions.
5. Differences Between Hank’s and Earle’s Balanced Salt Solutions
The main difference between Hank’s (HBS) and Earle’s (EBS) balanced salt solutions is the level of sodium bicarbonate. EBS contains higher levels (2.2 g/L) compared to HBS (0.35 g/L). Sodium bicarbonate requires high CO2 levels to maintain pH. In air, EBS becomes alkaline, while HBS becomes acidic in CO2 incubators. Use EBS for tissues in CO2 incubators and HBS for washing tissues stored in cell culture media.
6. Impact of pH on Cell Growth
Most cells grow best at pH 7.2-7.4. Deviation from this range can harm cell growth. Primary cells are less tolerant of pH changes, while immortal cell lines are more resilient. Generally, cells are more acid-tolerant than alkaline. Freshly prepared media may have a pH rise of about 0.2 after filtration through a 0.10um or 0.22um membrane.
7. Optimal CO2 Concentration for Cell Culture
Most culture media use the HCO3-/CO32-/H+ buffer system. The concentration of NaHCO3 in the medium determines the required CO2 concentration. Media with 3.7 g/L NaHCO3 need 10% CO2, while those with 1.5 g/L NaHCO3 require 5% CO2.
8. Benefits of Adding HEPES to Media
HEPES is a strong buffer that maintains stable pH between 7.2 and 7.6, even under open culture conditions when CO2 levels change rapidly. It is particularly useful for clonal cultures and primary neural cell cultures.
9. The Role of NaHCO3 in Media
NaHCO3-CO2 buffer system helps maintain pH balance in cell culture media. In open culture systems, CO2 produced by cell metabolism escapes, and the CO2 concentration in the incubator is adjusted to maintain equilibrium with NaHCO3 in the media.
10. Non-Essential Amino Acids (NEAA) in Media
NEAA mixtures, containing several non-essential amino acids, are added to the base media to support cell growth. For example, NEAA added to MEM includes L-Alanine, L-Asparagine, L-Aspartic Acid, L-Glutamic Acid, L-Glycine, L-Proline, and L-Serine.
11. Calcium Chloride in Osteoblast Culture
Calcium chloride can be toxic to osteoblasts, so it is excluded from osteoblast culture media.
12. Media Without Phenol Red
Phenol red is a pH indicator in culture media, turning red at neutral pH, yellow at acidic, and purple at alkaline pH. While it doesn’t affect the quality of biological products, phenol red can be omitted in serum-free media to avoid interference with cell growth assays. It can mimic steroid hormone action, so phenol red-free media are preferred for culturing hormone-sensitive cells.
13. bFGF and EGF in Cell Proliferation
Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are critical for stimulating cell proliferation and differentiation. bFGF binds to receptors on the cell membrane to promote gene expression related to growth, while EGF stimulates epithelial cell growth and repair. Both are added to media to enhance cell proliferation.
14. Insulin in Cell Culture
Insulin regulates metabolism, protein synthesis, and cell growth. It interacts with insulin receptors to mediate metabolic and growth-promoting effects. In serum-free media, insulin is added to maintain cell growth and regulate glucose, amino acid, and fatty acid uptake and utilization.
15. Vitamin A-Free B-27 Additive
The custom B-27® additive without vitamin A (retinol) is ideal for stem cell cultures, preventing the differentiation-inducing effects of retinoic acid. This 50x liquid additive can be combined with Neurobasal® or Neurobasal®-A media for neural cell cultures without feeder cells.
16. Antibiotics in Cell Culture
Antibiotics, such as penicillin-streptomycin solution (100x) and penicillin-streptomycin-amphotericin B solution (100x), prevent microbial contamination in cell cultures. The appropriate concentration of antibiotics ensures healthy cell growth by protecting against bacteria, fungi, and yeast.
Conclusion
Cell culture media provide essential nutrients and a living environment for cell growth and proliferation. Understanding the roles of various additives enhances cell culture efficiency and ensures optimal conditions for biological and medical research.