Cell Culture Media

Cell Culture Media is arguably the most important component in the culture beyond the type of cell used. This is a liquid or gel that is said to contain all the various vitamins and micronutrients needed for the cell to survive. They are typically listed in studies under the names Dulbecco’s Modified Eagle’s Medium (DMEM) or Minimal Essential Medium (MEM). There are many different kinds of media such as natural, artificial, chemical, serum, serum-free, protein-free, etc. so selecting the right type of media is considered crucial.

So what exactly does the cell culture media typically consist of?

“Cell culture media are comprised of a combination of compounds and nutrients designed to support cellular growth. Common components of cell culture media include:

  • Amino acids: Every cell culture media contains a mixture of amino acids, the building blocks of protein. Both essential and nonessential amino acids may be used to boost cell viability and growth. 
  • Vitamins: Vitamins are included to facilitate cellular growth and proliferation. Serum is used as the source of many vitamins in serum-containing media, but vitamins must be added to serum-free media. 
  • Carbohydrates: Carbohydrates provide an energy source for living cells. Glucose is commonly used, but other carbohydrates, such as galactose, fructose, or maltose, are available. 
  • Inorganic salts: Inorganic salts are needed to regulate membrane potential and osmolality.
  • Basic and trace elements: Cells need elements like iron, potassium, magnesium, and zinc to grow. 
  • Serum: Serum contains growth factors and inhibitors, hormones, protease inhibitors, chelators, amino acids, carbohydrates, lipids, vitamins, trace elements, minerals, and more that are needed for cellular growth. Bovine serum is commonly used. 
  • Hormones: Certain hormones may be added to influence cell function, growth, and proliferation. 
  • Buffering systems: Buffering systems regulate pH.
  • Supplements: Supplements like hormones, enzyme inhibitors, and trace elements are sometimes added to cell culture media that cater to the cell-type and research goal.
  • Antibiotics: Antibiotics are added to cell culture media to inhibit fungal and bacterial growth. Antibiotics are best suited for serum media thanks to proteins that bind some of the antibiotic load. In contrast, cells in serum-free media are at a greater chance of antibiotic toxicity.”  


It is clear that the media consists of many different compounds said to keep the cell alive and aid in the growth of any “viruses” present. However, there are many issues related to the cell culture media that can influence the outcome of the culture and the results obtained. These include: the unknown and variable composition of the vitamins/micronutrients within the media, the unknown interactions between the various components in the media and the stability of the cell, the inclusion of antibiotics and fetal bovine serum in many media, etc.

For instance, from the same source quoted above regarding the makeup of cell culture media, come these admittances regarding serum media, the most commonly used being Fetal Bovine Serum:

But there are downsides to serum media. When it comes to its drawbacks, serum media:

  • Lack compositional uniformity, making it poorly suited for large-scale experiments. 
  • Contain a mixture of compounds, some of which can be harmful to or inhibit the growth of certain cell types.
  • Have a greater risk of contamination when compared to artificial media.
  • May complicate the isolation of cell culture products.”

These are but a few of the issues and more are outlined in the articles below:

Commercially available cell culture media contains buffers, inorganic salts, glucose, amino acids,vitamins and numerous bioactive compounds but the levels of each may differ substantially. For instance, the concentration of glucose in the media varies between 5.5 mM and 25 mM (Minimum Essential Medium (MEM) vs. Dulbecco’s Modified Eagle’s Medium (DMEM)), and the presence of ferric nitrate in DMEM and copper, iron and zinc sulphate in F12 medium, might interact with specific metal dependent enzymes of importance in cellular reactions.”

“Immortalized cells are a commonly used model system in biomedical research due to its simplicity, availability and high throughput characteristics. However, the composition of cell culture medium used varies extensively and may affect the results from biochemical, toxicological and pharmacological studies substantially depending on the experimental conditions.”

“However, the differences demonstrated in cell proliferation may not only depend on the different levels of glucose and cysteine since the media differ greatly in other components as demonstrated by iron, for example.”

A change in phenotype due to medium composition is a serious effect that could profoundly change the outcome of any pharmacological and mechanistic studies. These observations must therefore be carefully taken into consideration when interpreting any data from cell experiments.”


From this article, it is clear that the media:

  • contains substantially differing levels of micronutrients
  • may effect the results of experimental conditions due to varying composition

These next two sources also provide evidence that the makeup of the various compounds in the media can have an affect on the results obtained from the cell culture experiment:

15 things about cell culture you might not know

“Components of culture medium can interact with each other and influence cultured cells.”

When you are trying to optimize your culture medium, consider this: Individual components of the medium do not act alone. Components can interact, and their effects on cells are not always predictable. This is particularly important when replacing animal sera in culture media. You might need to use mathematical algorithms to optimize the combination of multiple compounds and to establish the best conditions for cellular growth (Yao and Asayama, 2017Kim and Audet, 2019).”

15 things about cell culture you might not know

The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells’ responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients’ roles at a molecular level and influence on the genomic stability of cells are still needed.”

“The composition of these media includes certain vitamins and minerals, but unfortunately, in many common culture media, the only source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Moreover, the appropriate proportion of micronutrients is not always provided because the precise composition of each batch of FBS is in fact extremely variable [34].”

“Certain micronutrients, such as calcium, folate, magnesium, and iron, have been reported as key elements in cellular processes, including the proliferation, survival, and even differentiation of cell cultures [3538]. However, the particular concentration of micronutrients in a culture as well as the cell type may trigger different responses. Further studies of micronutrients’ roles at a molecular level and influence on genomic stability are still required.”

“Table 2 presents interesting data regarding the micronutrients that may interfere with genomic stability and the micronutrient concentration values found in typical cell-culture media, FBS, and human serum. Unfortunately, data are not available for all of the micronutrients in the media, and even the proportions of micronutrients in FBS, as an organic product, are not all well characterized. Additionally, as demonstrated by Bryan et al. [34], the concentration of many micronutrients in FBS can vary significantly between batches.

Although cell-culture media attempt to provide an environment similar to the in vivo milieu of cell development, there is an evident imbalance of micronutrients between the media and human serum. Certain micronutrients are present in these media at concentrations higher than those found in human serum (e.g., vitamins B7 and B12), whereas other nutrients are present at significantly lower concentrations than in human serum (e.g., iron and zinc).”

“Cells are typically maintained at an appropriate temperature and CO2 concentration (usually 37°C and 5% CO2 for mammalian cells) in an incubator. Beyond these parameters, the most commonly varied factor in culture systems is the growth medium. The recipes for growth medium can vary in pH, glucose concentration, growth factors and the presence of other nutrients and micronutrients.”

“Bryan et al. [34] stated that one of the major obstacles to obtaining human cells of a defined and reproducible standard, and thus suitable for use in medical therapies, is the routine necessity of supplementing cell-culture media with FBS. In this study, FBS variants were evaluated, in terms of both elemental (micronutrient) composition and the variants’ effects on the expression of a group of proteins associated with the antigenicity of primary human umbilical vein endothelial cells (HUVECs). A combination of inductively coupled plasma mass spectrometry (ICPMS) and flow cytometry was used to achieve these experimental objectives. Statistically significant differences in antigenic expression during cell culture were demonstrated for a set of trace elements in FBS (e.g., lithium, boron, magnesium, phosphorus, sulfur, potassium, titanium, vanadium, chromium, manganese, iron, copper, zinc, gallium, and selenium). The lack of reproducibility and the variation in protein expression in the primary human cells was attributed to the FBS supplementation.

Culture conditions for cell lines are known to affect gene expression [154156], while stem cells grown in different types of serum exhibit variable differentiation and proliferation characteristics [157158] the same cell line, if cultivated in different conditions, can present different phenotypes. Nevertheless, the cellular requirement for a specific micronutrient is directly correlated with the cell type, the rate of cell grow, and the stage of cell differentiation. In light of this, it is important to observe that minimal attention has been dedicated to the composition of FBS and the micronutrient supplementation of media in cell cultures or the fact that micronutrients can influence the viability and genomic stability of cultured cells.”

“Even though there are some highly enriched media available as basal media for serum-free cell culture, like Medium 199 or Ham F-12 nutrient mixture, the most common source of micronutrients currently used in cell cultures is still FBS. The limitations of FBS in providing adequate micronutrient concentrations have been analyzed and described in the literature [34]. Given that cell- and tissue-culture models are generally important in scientific research, the development of standards in vitro methods is mandatory. “

At the very least, an evaluation of FBS composition, in terms of micronutrients and possibly other factors, should be strongly considered in the laboratories that focus on in vitro studies. Knowledge of the micronutrient composition of FBS may help to minimize the bias in experimental results. However, maintaining both successful and consistent cell cultures can be difficult, as FBS is a complex natural product and may vary between batches, even if obtained from a single manufacturer. More specifically, the quality and concentration of both bulk and specific proteins in cell cultures can affect cell growth [210]. Adjusting the in vitro micronutrient levels to physiological values will guarantee a better environment for cell development, mimicking the in vivo milieu.

Further studies on the effects of micronutrients on cell viability, proliferation, and stability, as well as gene expression and integrity are still required, but the information already available is a sufficient call to action. As mentioned by Ferguson and Fenech [141], most investigations have been limited to studying the effects of single micronutrients and have not considered genetic consequences. Thus, there is an important need for studies that also examine nutrient-nutrient and nutrient-gene interactions. Determining the physiological range of such significant micronutrients as iron and then adjusting the concentrations currently found in cell-culture media may be beneficial for in vitro assays. More specifically, the viability and genomic stability of cell lines and primary cultures may be improved. Depending on the cell type (primary, immortalized, tumor, or normal) and origin (lung, hepatic, neural, or other), the requirement for a micronutrient may vary widely, so this subject should be carefully evaluated. Finally, the form of the micronutrient used in supplementation media may also influence experimental results.


In Summary:

  • Media contains components that interact with each other and can influence the cells
  • The effects are not always predictable
  • Media lacks compositional uniformity
  • Fetal bovine serum is a main component of many media
  • Minimal attention has been paid to the composition of the media as well as the interaction on viability/genetics of cells
  • Media can influence and interfere with genomic stability
  • Each batch of media is variable
  • Concentration of micronutrients may trigger different cellular responses
  • Data is not available for all the micronutrients in media nor how they interact/affect the cells
  • There is an imbalance in micronutrients and human serum
  • Media recipes vary in Ph, glucose concentrations, growth factors, and the presence of other nutrients/micronutrients
  • Culture conditions affect gene expression
  • Further studies on the effects of media on cell cultures are needed
  • The form of the micronutrients may influence experimental results

It is clear that there are numerous issues with cell culture media, even in the absence of antibiotics and FBS which by themselves can have profound impacts on the cells and the outcome of a culture. There are too many unknowns about the makeup of the media and the interactions between the various components. With so many known and unknown variables/issues, there is no way to be confident that cell culture media has no impact on the end result of any culture/experiment. In fact, it is a guarantee for media to have an impact. The problem is that the extent of this impact is unknown.


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