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The team at Captivate Bio has been supporting the manufacturing and development of various types of classical media and stem cell media for the life science industry for over 20 years. During this time, we have seen advancements in media optimization and the emergence of specialty media for specific cell types with applications ranging from stem cell research to cell banking and vaccine production.
Culturing cells is one of the most common techniques used in the life sciences today and cell culture media itself is a critical tool for maintaining and growing healthy cells in vitro. Understanding how cell culture media and each component impacts your cell line is critical to selecting the ideal environment. These basic media components include the base, salts, buffers, carbohydrates, amino acids, vitamins, antibiotics, proteins, serum, and other elements.
Here we break down a few of the basic key components included in cell culture media in order to understand the importance of each and how they may impact your cell type.
Many of the classical basal media being used today include DMEM, MEM, and RPMI 1640, which are historical published formulations that are used as the base formulation for many of the popular chemically defined media we see today. Each of these basal media were developed to support a particular cell line or culture condition and over time ingredients (or components) have been added or removed to create many different options of each basal media we see today. The main differences between an RPMI or DMEM are the identity and quantity of buffers, salts, glucose, vitamins, and growth supplements included in each media.
A good example of a basal media developed over time is DMEM/F12. This basal media has become a popular choice for culturing stem cells due to the mixture with Hams F12, a complex medium with higher amino acids and vitamin concentrations. Other examples include RPMI 1640 manufactured with or without L-glutamine or glucose for culturing cells in cancer research applications.
Depending on your cell type, the cell culture media you select must meet the basic nutrient needs of the cells in culture. For example, most mammalian cells grow well at a pH of 7.4, and maintaining this pH is critical. When using vented flasks and culture dishes, the CO2 gas in the atmosphere dissolves into the cell culture medium and establishes equilibrium with HCO3-. Because CO2 is acidic, the pH of the medium is lowered. Although salts and amino acids within the medium can provide some buffering capacity, additional buffering compounds are added to ensure that a proper physiological pH is maintained. Below are a few key components added to media to aid in buffering.
Sodium Bicarbonate (NaHCO3) serves as a pH buffer in cell culture media, especially in CO₂ incubators where it interacts with dissolved CO₂ to maintain a physiological pH (7.2–7.4). It also provides ions which are essential to supporting cell metabolism. As a rule of thumb, 1.5 g/L of NaHCO3 is needed to keep the medium at pH 7.4 when cultured in 5% CO2 incubator. In a controlled CO₂ environment, alternative buffers like HEPES may be used in experiments conducted outside incubators. Figure: CO2 in cell culture media yields acidic conditions. Sodium bicarbonate helps buffer this reaction.
HEPES is a zwitterionic organic buffer that is also used to maintain physiological pH of cell culture media. HEPES is recommended when the cell culture system is very sensitive, increasing the buffering capacity and stabilizing the pH within the range of 7.2 to 7.6, and is not dependent on CO2 levels.
Phenol Red is commonly included in cell culture media as a visual pH indicator. Media containing phenol red will appear red at a pH of 7.2-7.4 which is optimal for most mammalian cells. A pH range of <6.5 or bright yellow may be acidic with potential contamination or bacteria buildup. And a pH range of >8.0 or purple may be contaminated or with possible CO2 loss in the media. Media that appears bright yellow or purple is generally unsuitable for sensitive cell types and visually indicates that the culture requires immediate attention (medium exchange, passaging, or incubator gas adjustment, etc.).
There are a few potential drawbacks of phenol red as it can interfere with assay results or introduce cell stress. Choosing a phenol-red free cell culture media is important for hormone-sensitive studies, fluorescence assays, or working with sensitive cell lines.
Cells require nitrogen to build nucleotides, vitamins, and amino acids to aid in cell growth, protein synthesis, and metabolic functions. There are two types of amino acids: essential and non-essential. Cells cannot synthesize essential amino acids, and they must get them from the media (leucine, lysine, methionine). Cells can naturally synthesize non-essential amino acids (NEAA), however, supplementing them in the media stimulates proliferation and prolongs the viability of the cells in culture (alanine, glutamine, serine). Some amino acids serve as an alternative energy source to glucose while supporting viability and proliferation. Here we cover a few key components of amino acids in cell culture media.
Glutamine is an essential amino acid that is a required additive to most cell culture media, facilitating the storage and transfer of nitrogen to the cells in culture. Glutamine is also one of the most readily available amino acids for use as an energy source when glucose levels are low and energy demands are high, especially for rapidly proliferating cell types or cells cultured under hypoxic conditions, where glucose metabolism is less efficient.
However, when added to media, L-glutamine can be quite unstable thus why many classical media containing L-glutamine may have a shelf-life of 12 months or less when stored at 4°C. The L-glutamine may non-enzymatically degrade into toxic ammonium and pyroglutamate by-products which leads to a short shelf-life and why stock solutions of L-glutamine must be stored frozen at -20°C. There are now stable L-glutamine products on the market that may extend the life of your media past 12 months or stored in various conditions including room temperature.
L-Alanyl-L-Glutamine is a dipeptide that is stable in cell culture medium over longer periods of time, even at 37°C. In order to combat the rapid breakdown and stability within cell culture media, L-glutamine may be supplemented with L-Alanyl-L-Glutamine. The strong bonds within L-alanyl-L-glutamine stabilize the compound from degradation, while the L-glutamine itself is still readily accessible to the cells. Supplementing media with L-alanyl-L-glutamine can extend the shelf-life of the media at 4°C and greatly reduce the problems associated with the breakdown of glutamine into ammonia waste.
Antibiotics are often used by labs to control the growth of bacterial contaminants that can occur in the nutrient-rich cell culture environment. Other than primary cell isolation, the use of antibiotics is not necessary or encouraged in cell culture media. In fact, routine use of antibiotics can hide low levels of bacterial contamination, generate resistant bacteria strains, mask the effects of mycoplasma contamination, and even interfere with cell metabolism.
However, you may look to add antibiotics to media that is highly susceptible to contamination. Adding antibiotics can act as a safeguard, especially in long-term cultures or when working with multiple samples. Add antibiotics only when necessary, maintain proper aseptic techniques, and regularly test for mycoplasma contamination as most antibiotics do not eliminate mycoplasma in cultures.
Common antibiotics include:
Growth factors, cytokines, interleukins, chemokines and other hormones like insulin are the chemical messengers of cells. They are small protein molecules naturally secreted by cells that induce a physiological effect (proliferation, growth, or differentiation) on the cells receiving the signal. Depending on your cell type, your cell culture media may need specific growth factors to maintain or direct the cell’s health, proliferation, or differentiation. Here we highlight a few examples of protein additives.
Growth Factors added to cell culture media are generally recombinant proteins and can be synthesized in multiple types of cells and species, including E. coli, CHO, and human lines. Always use growth factors with high biological activity (ED50), high purity, and proper protein folding and other post-translational modifications for the best results. Growth factors and cytokines are stable when stored as lyophilized powders but have a short shelf-life once added to media. Many complete media on the market have containing proteins have a shelf-life of 18 months or less when stored at -20.
Common growth factors added to media include:
Cytokines are a large group of low molecular weight proteins, polypeptides or glycoproteins that are secreted by various immune cells including macrophages, lymphocytes and mast cells, as well as other cell types such as endothelial cells. They play important roles in regulating cell growth, differentiation, and activation, and are involved in many aspects of both innate and adaptive immune responses. Cytokines include interleukins, chemokines, interferons and other signaling molecules. However, there are some cytokines that are considered growth factors due to the signaling pathway.
Please note that the terms growth factors, cytokines, interleukins are often used interchangeably.
Common cytokines or interleukins added to media include:
Today, there are many options in selecting the best stem cell culture medium for your cell type. Options include making your own media in the lab, scaling up with a reliable partner, purchasing complete commercially available media that are all convenient alternatives that can guide you in media selection.
When looking at an off-the-shelf complete medium, this implies that all the required components are included in the cell culture media and that formulation has been validated with specific cell types and/or specific applications. Using pre-validated complete media can eliminate the variability between batches caused by inconsistencies in the ingredients used or the preparation procedure itself. Whether making media from scratch or using pre-made complete media, it is always important to monitor the cells in culture to ensure the media is performing optimally for your specific cells and application.
To learn more about how Captivate Bio can help with your cell culture media selection or to request a quote, contact us today.
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