Anyone who has performed mammalian cell culture will recognize the typical media recipe: 1 bottle of DMEM, 10% serum, a few other magical ingredients, throw it together and put it on your cells to keep them alive and happy. However, do you know what each ingredient does? Let’s look at the common ingredients in cell culture media and break down their roles:
Sweeten It Up
Every growth media requires a carbon source that cells can metabolize. This is often glucose, but it can also be other sugars such as galactose, hexose, fructose, or other carbon sources (e.g., pyruvate or glutamine). Glucose is commonly added at a final concentration of 5.5 mM, which is approximately equal to the glucose levels found in human blood. However, you can buy media with wide ranges of glucose concentrations to suit your specific needs. You can even buy glucose-free media and then customize the concentration yourself to simulate high-sugar diabetic conditions or to study low-glucose stress responses.
Buffer It Out
Cells require a very specific pH from 7.2 to 7.4, otherwise they cannot perform their cellular functions optimally to grow and survive. In incubators with high carbon dioxide levels, CO2 reacts with water to produce hydrogen ions that turn the media acidic. To counteract the acidity generated by CO2 and maintain the optimal pH in culture, most media types use a chemical buffering system. However, some buffering methods interfere with cell growth.
The most common buffer system for mammalian cells with minimal biological impact is sodium bicarbonate. Sodium bicarbonate reacts with the hydrogen ions generated by CO2 and sequesters them to maintain pH. One disadvantage with sodium bicarbonate, however, is that it doesn’t buffer optimally at physiological pH. To get around this issue, researchers often use alternatives, such as HEPES, to increase the buffering capacity of certain media types.
Because maintaining pH is so critical to cell culture, many media formulations include a pH indicator called phenol red that turns yellow in acidic conditions (<6.8), and pink in basic conditions (>8.2). While generally not a problem, phenol red can mimic estrogen and, therefore, is commonly excluded when culturing cell types that express the estrogen receptor. Media without phenol red is also available for colorimetric assays, such as the MTT proliferation assay, where the color spectrum of phenol would likely interfere with spectrophotometric readouts and, thus, confounding the results.
An essential component for many culture systems is serum, commonly fetal bovine serum—better known as FBS. Derived from blood, serum is an undefined mixture of sugar, salts, lipids, growth factors, and much more. Because of its biological source, it can be difficult to maintain reproducibility, as each batch of serum is unique. To circumvent this, some labs use chemically defined serum replacements, while other labs buy entire batches of one particular serum lot to ensure consistent results—at least until that lot runs out!
Metabolites, Vitamins, and Minerals, Oh My!
If you look at the list of ingredients in DMEM, the majority fall into three major categories: amino acids, vitamins/cofactors, and inorganic salts (including calcium and magnesium). Many of these components are common in all cell culture media, and the exact combination and concentrations have been optimized for cell growth. Despite this, many labs choose to supplement their media with additional non-essential amino acids to prevent cells from using glucose or glutamine for their synthesis. Additionally, some companies produce magnesium and calcium-free media for specific studies; so make sure you check the components carefully when you order!
Many labs include antibiotics in their cell cultures to prevent unwanted bacterial or fungal growth, even when practicing aseptic technique. The most popular choice of antibacterial supplement is a combination of penicillin and streptomycin (pen/strep) at a final concentration of 50–100 IU/mL penicillin and 50-100 µg/mL streptomycin in complete cell culture. However, some labs choose not to use antibiotics as they can 1) have unwanted effects on your cells, or 2) can mask low levels of bacterial contamination. Depending on your needs, you can decide whether or not to include them in your media.
That was it for this time! Next time you make media, pay extra attention to which bottle of DMEM you add and which supplements your cells need. Knowing these things will hopefully result in happy healthy cells, putting you on the right track for great experimental results. It will hopefully also give you a deeper insight into the inner workings of your cell culture system.