Cell Culture Basics: Stem Cell Media – The “What” and “Why”

A guest blog by Lia Kent, Scientific Training, Biological Industries (BI)


For over 30 years, Biological Industries has been developing specialized cell culture media for many cell types, including primary cells and stem cells. Over time we have seen a significant evolution of stem cell media as well as our ability to understand, evaluate, and optimize cell culture conditions. Cell culture is one of the most common and complex techniques used in the life sciences, and the media itself is a critical element for maintaining healthy, proliferating stem cells in culture. When you break it all down, all stem cell media essentially contain the same basic components: a basal medium, buffer system, glutamine, serum (or serum alternative), specific growth factors, and additional supplements. Here we look at a few of the main media components to better understand their importance and influence on cell cultures and how working with a highly pure and defined stem cell medium is key for regenerative medicine applications.

Basal Media

Classical basal media (DMEM, MEM, RPMI, etc.) are chemically defined formulations, each developed to support a particular cell line or culture condition. The main differences between the various classical basal media are the identity and quantity of buffers, salts, and growth supplements.

Many of the basal media were originally developed using mouse fibroblasts, HeLa, or CHO cell lines, and modifications over time have established basal media suitable for a wide range of cell types. DMEM/F12 is a popular stem cell basal medium, and a good example of this type of modification, as it is a 1:1 mixture of a complex medium (Ham’s F12) and a medium with higher amino acid and vitamin concentrations (DMEM).

Interestingly, the names of classical basal media represent the researcher or the institute who developed them. (In the examples below, you can also see how basal media can be modified over time by multiple researchers.)


  • RPMI = Roosevelt Park Memorial Institute
  • BME = Eagle’s Basal Medium (Eagle = developing researcher)
  • MEM = Modified Eagle’s Medium (Eagle = developing researcher)
  • DMEM = Dulbecco’s Modified Eagle’s Medium (Dulbecco = developing researcher)


Stem cell media must match not only the basic nutrient needs of the cells in culture, but also be appropriate for the culture conditions being used. Most mammalian cells grow well at a pH of 7.4, and maintaining this pH is critical for stem cell cultures. 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.

Sodium Bicarbonate

To stabilize changes in pH within a CO2 incubator, sodium bicarbonate (NaHCO3) is a non-toxic natural buffer commonly included in stem cell medium. As a general 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.

H2O + CO2   ↔   H2CO3     ↔   H+ + HCO3¯

NaHCO3   ↔   Na+ + HCO3¯

Figure: CO2 in cell culture media yields acidic conditions. Sodium bicarbonate buffers 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

In addition to CO2 levels, cell metabolites, media additives, and a variety of other factors can also impact the pH of stem cell media. For this reason, phenol red is commonly included in stem cell media as a visual pH indicator. Media containing phenol red will appear red at a pH of 7.4, and will range from bright yellow to deep purple as the pH fluctuates. Media that appears yellow or pink/purple is generally unsuitable for stem cells, and indicates that the culture requires immediate attention (medium exchange, passaging, incubator gas adjustment, etc.). Choose a medium without phenol red if your cells are particularly estrogen-sensitive, as this compound mimics the actions of some steroid hormones.


Figure: Phenol red pH chart -- Media containing phenol red will appear red with a pH of 7.4, and will range from yellow to purple as the pH changes. Making a pH standard for a particular basal medium can be helpful and used in the lab as a more accurate visual reference.
Figure 1: Phenol red pH chart — Media containing phenol red will appear red with a pH of 7.4, and will range from yellow to purple as the pH changes. Making a pH standard for a particular basal medium can be helpful and used in the lab as a more accurate visual reference.



Cells require nitrogen to build nucleotides, amino acids, and vitamins. L-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 dissolved in liquid media, free L-glutamine is quite unstable. At temperatures above 4°C, the L-glutamine within cell culture media non-enzymatically degrades into toxic ammonium and pyroglutamate by-products. This short half-life is why stock solutions of L-glutamine must be stored frozen at -20°C, and why L-glutamine should be added to freshly prepared media just prior to use.


In order to combat the rapid breakdown and increase glutamine stability within cell culture media, L-glutamine dipeptides are often substituted. L-alanyl-L-glutamine, for example, is a dipeptide that is stable in cell culture medium over longer periods of time, even at 37°C. 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 stem cell 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.

Figure 2
Figure 2: Chemical structure of L-glutamine and the stable dipeptide, L-alanyl-L-glutamine. Stem cells in culture can easily break the stabilizing bonds in L-alanyl-L-glutamine to retrieve and use L-glutamine as needed.

Growth Factors and Supplements

Growth Factors

Growth factors, cytokines, and chemokines 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. Each stem cell type requires media supplemented with specific growth factors in order to maintain or direct the culture’s health, proliferation, and differentiation. For example, bFGF (fibroblast growth factor-basic) is the key growth factor involved in maintaining pluripotency for human embryonic stem (ES) and induced pluripotent stem (iPS) cell cultures, while LIF (leukemia inhibitory factor) is the main regulator for mouse ES and iPS cells.

The quality of the growth factors used in stem cell media can have a profound impact on the culture. Growth factors used in 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. To avoid degradation of these components, prepare small aliquots of concentrated stock solutions, and add them to media just before use.

Amino Acids

In addition to essential amino acids supplied in the basal medium and supplemented glutamine, non-essential amino acids (NEAA) are also often added to culture medium to stimulate proliferation and prolong the viability of the cells in culture. Although cells can naturally synthesize NEAA, supplementing them in the media reduces any potential side-effects that can affect cell growth when amino acid levels are low.


Supplemented 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 stem cell culture. 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.

Serum and Serum Alternatives


Most mammalian cell cultures require the addition of animal or human serum to the media. The basic definition of serum is “the liquid component of clotted blood.” Serum for cell culture is a complex organic mixture of proteins, peptides, growth factors, amino acids, lipids, carbohydrates, hormones, vitamins, and many other components. Some cell cultures depend on serum to provide not only nutrients, but compounds that promote cell proliferation and attachment.

Stem cell media that includes serum commonly contains between 10 to 20% fetal bovine serum (FBS).   Although serum used in stem cell media generally undergoes numerous modification and purification steps, the end product will always hold the same key disadvantages for stem cell applications, notably that the material is undefined, highly variable between batches, and derived from an animal source.

Serum-free Media

Serum-free (SF) media allow researchers to culture stem cells in the absence of serum. The main advantage of serum-free stem cell media is the consistent and reproducible experimental results from a more pure media formulation. In general, serum-free media is considered defined media, containing albumin, insulin, selenium, and transferrin proteins in place of FBS. Defined serum-free media can include serum-derived proteins, however, such as human serum albumin (HSA).

It is important to know that stem cells can be much more sensitive when cultured in a serum-free environment. This can be especially noticeable with regard to morphology, cell density, proliferation rates, and sensitivity to mechanical and chemical stresses, including dissociation enzymes and antibiotics. If using antibiotics in serum-free media, for example, it is generally best to use a final concentration of 1/5th to 1/10th of what is normally used for serum-containing media.

Figure 4

Defining Stem Cell Media For Regenerative Medicine

Culturing stem cells for downstream therapeutic or clinical applications requires strict adherence to cGTP and cGMP guidelines, as well as the use of a defined culture system. This includes not only the cell culture media, but also attachment substrates, dissociation solutions, cryopreservation solutions, and differentiation systems. The field generally uses standard terms to define the level of purity of the media, though often these terms are misunderstood.

A chemically defined (CD) medium is one in which all of the chemical components of the medium and their exact concentrations are known. In order to be chemically defined, a medium must contain recombinant versions of the albumin and other required factors. A chemically defined medium would use recombinant albumin protein in place of human or bovine serum albumin, for example.

Xeno-free (XF) media contain no animal-derived components, but may contain human-derived components. The word “xeno” means “alien” or “strange” – or in this case, “non-human”. Xeno-free media can be classified either as defined (if all proteins are human-derived) or chemically defined (if all proteins used are human recombinant proteins).

Animal component-free (ACF) media is one more step forward in improving the definition of stem cell media. Animal component-free media contain only synthetic or recombinant factors, and often this definition encompasses the materials used to produce the media components as well as the media itself.


Figure 5 high res Figure 5 arrow

Serum → Bovine Serum Albumin → Human Serum Albumin → Recombinant Human Albumin

Media Validation

Today, high-quality complete stem cell culture media are commercially available to researchers, such as NutriStem® MSC Medium or NutriStem® hESC Medium, which offer a convenient and consistent alternative to preparing media in the lab. A complete medium implies that all of the required components are included in the solution and that the medium 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.

Cell morphology is one of the first and simplest ways to evaluate the overall health of the cultures, since changes occurring in stressed or spontaneously differentiating cells can often be observed under the microscope. Proliferation and cell growth are also key performance parameters to note. Poor cell proliferation can be a result of nutritional deficiencies or other environmental stresses that can inhibit cell division. Cell viability after passaging, thawing, and other procedures can be assessed using a trypan blue dye exclusion assay. Expression of appropriate surface and/or nuclear markers should also be monitored over time, either by flow cytometry or immunocytochemistry. Directed differentiation assays are performed in order to show that the cells retain their appropriate differentiating potential in the media. In addition, chromosomal analysis should be assessed with new media formulations or other changes in the culture system to demonstrate that a normal karyotype is retained during long-term culture.

Stem cells hold great potential for groundbreaking applications in regenerative medicine and to advance our understanding of human biology. Successful stem cell cultures require both optimized protocols and high-quality reagents. Increasing the chemical definition of stem cell media, along with high-quality, cGMP-grade reagents will improve consistency and validity of experimental results, and ultimately enable the reproducible generation of quality-assured stem cells for the development of breakthrough clinical uses.

Don’t miss the next in the series: Cell Culture BasicsMycoplasma 101 – A practical guide to prevention, detection and elimination of mycoplasma contamination

About Biological Industries
Biological Industries is one of the world’s best kept secrets. With over 30 years’ experience in cell culture media development and manufacturing, we collaborate with and supply reagents to many of the fields’ leading academic institutes and biotechnology companies. Our products range from classical cell culture media to serum-free media, supplements, and reagents for Stem Cell Research and potential Cell Therapy applications, cytogenetic reagents, and molecular biology kits. We are committed to a Culture of Excellence through our advanced manufacturing and quality control systems, superior regulatory expertise, in-depth market knowledge, and extensive technical customer support, training, and R&D capabilities.

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