FAQs of Culture Medium
What is the composition of the culture medium? What is the purpose of each of them?
To support cell growth, all media contain the basic nutrients, water, carbon, nitrogen, vitamins, amino acids, fatty acids, trace elements, phosphates. The medium contains more than 70 nutrients and the proper combination of the various components becomes the key to optimization. The basic components of the medium include:
| Components | Role | Major categories | Optimization method |
|---|---|---|---|
| Sugars | The main energy substance and carbon source | Mainly glucose | Since glucose as a six-carbon sugar is easily metabolized to lactic acid, five-carbon sugars such as fucose, galactose, mannose, etc. are often used instead to reduce lactic acid production |
| Amino acids | Major source of nitrogen | Divided into non-essential and essential amino acids | For different expression strains, reasonable optimization of amino acid ratios can effectively increase the live cell density and expression of recombinant proteins |
| Vitamins | Important active substance for maintaining cell growth | Divided into water-soluble and fat-soluble vitamins | The cell culture medium mainly contains B vitamins. The vitamin requirements of different cell lines vary greatly and need to be optimized |
| Lipids | Main component of cell membrane, involved in signal transduction | Divided into saturated and unsaturated fatty acids | The addition of linoleic acid, oleic acid, ethanolamine and choline can promote cell proliferation |
| Trace elements | Major component of cellular metabolism, regulates enzyme activity | The main trace elements are: Cu, Fe, Mn, Mo, Ni, Se, Si, Zn | Fe is involved in cell replication and is an essential element; Se is an important component of protein and can improve the antioxidant capacity of cells; Zn can replace growth factors and play a role in stimulating cell growth |
How to choose the culture medium?
- Choose the medium according to the characteristics of the cell line and the needs of the experiment. For example, for cell hybridization and gene transfer experiments, IMDM (Iscove's Modified Dulbecco's Medium) can be selected.
- Use a variety of medium to culture the target cells and observe their growth status, which can be judged by growth curve, colony formation rate and other indicators, and choose the best medium according to the experimental results, which is the most objective method.
Can liquid culture media be stored at -20°C?
No! Because at -20℃, the salt in the medium is easy to precipitate out, and after the medium melts, some salt may not be able to redissolve, which leads to the reduction of the osmotic pressure of the medium, and when the cells are cultured, the cells are easy to rupture and die.
How long can I keep freshly prepared liquid culture medium?
It is recommended to store freshly prepared medium at 4°C, away from light, and try to use it within a week. The fresher the medium, the better.
Could the medium be stored for a long time after serum and antibiotics have been added to it?
Once serum and antibiotics have been added to fresh medium, it should be used within two to three weeks. This is because some of the essential components of the antibiotics and serum begin to degrade after thawing.
Do I have to add antibiotics to the culture medium?
Generally, no antibiotics should be added to the medium in the normal culture state, except for special screening systems. For example, in the yeast two-hybrid system (Y2H), Aureobasidin A can be used as a key component of yeast media, which inhibits yeast inositol phosphoryl ceramide (IPC) synthase and provides excellent screening results for drug discovery.
To prevent bacterial and fungal contamination, a penicillin-streptomycin solution can be added with a final concentration of 100 U/ml of penicillin and 100 μg/ml of streptomycin in the medium.
Why does the culture medium become darker red in color and more alkaline in pH when it is stored in 4°C?
When the medium is stored in 4℃, the CO2 in the medium will gradually escape, causing the medium to become more alkaline. The color of the acid-base indicator (usually phenol red) in the medium will also be darker red. The result of alkaline medium will cause cell growth stagnation or death. If the medium is alkaline, the pH value can be adjusted by introducing sterile filtered CO2.
How to maintain the pH of the medium?
A buffered system of balanced salt solution is used to maintain the pH range required for cell physiology. The four major cations in physiological solutions are Na+, K+, Ca2+, and Mg2+. All four ions are important for maintaining membrane potential and nutrient transport. The Na+ and K+ play a key role in determining osmotic pressure and maintaining osmotic balance. The Ca2+ and Mg2+ are important as substrates for cells to adhere to each other and serve as cofactors for enzymatic reactions.
The main anions in the equilibrium solution are PO43-, Cl- and HCO3-. These anions are used in the buffer system to maintain an optimal pH of 7.2-7.6 for the cells. Phosphate and amphoteric salts are generally chosen as buffer systems in the basal medium. When using a medium with NaHCO3, the optimal ratio of CO2 varies depending on the formulation of the medium. In general, for media containing 2-3 g/L of NaHCO3 at a temperature of 36-37°C and a controlled CO2 content of 6% in the incubator or shaker gas environment, the medium should achieve a pH suitable for cell growth.
How does the pH of the culture medium affect cell growth?
Since most cells have a pH value of 7.2-7.4, deviation from this range will have harmful effects on cells. The pH requirements of various cells are not exactly the same. Primary cell cultures are generally poorly tolerant to pH changes, while continuous cell lines are highly tolerant. However, in general, cells are more acid tolerant than alkaline. Therefore, the pH of the culture medium should be adjusted slightly to acidic. When the culture fluid is filtered through 0.1 μm or 0.2 μm membrane, the PH value will float upward about 0.2.
Why do some cultures not contain phenol red?
Studies have shown that phenol red can mimic the effect of steroid hormones (especially estrogen). To avoid steroid reactions, cell culture, especially for mammalian cells, is done with medium without phenol red. Because phenol red interferes with the assay, some researchers do not use phenol red-added medium when doing flow cytometry.
Is the amount of antibiotics used in serum-free culture the same as in serum culture?
When adding antibiotics to the serum-free medium, reduce the concentration used in the serum medium by at least 50%. This is because the proteins in the serum will bind and inactivate some of the antibiotics. In serum-free culture conditions, antibiotics are not inactivated.
What is the classification of serum-free media?
- Serum-free medium (SFM): Serum-free, replacing functional alternatives to serum, such as transferrin, trypsin, adhesin, etc.
- Protein-free medium (PFM): Generally refers to medium that does not contain high molecular weight proteins of animal origin, but may contain low molecular weight peptide fragments of proteolysis of plant origin. Protein-free media facilitate the isolation and purification of downstream cell products.
- Chemically defined medium (CDM): The chemical structure of all components of the medium is completely defined to ensure batch-to-batch consistency and the nature of the components is clear, which is conducive to metabolic studies of cell culture and large-scale production, as well as easier isolation and purification.
What are the advantages of serum-free media?
- Improved cell expression yield
- Easier downstream product isolation and purification
- Stable composition for mass production
- Improve R&D and production efficiency
- Reduced costs
- High specificity
- Avoiding serum toxicity to cells and serum-derived contamination
- Improve the accuracy of experimental results
- Reduced animal suffering and no ethical restrictions
