How to Select the Right Cell Culture Media

This article was published first in "Inside Cell Culture" , the monthly newsletter for cell culture professionals. Find more interesting articles about CO2 incubators on our page "FAQs and material on CO2 incubators" .

• An introduction to cell culture

• The nutritional requirements of different cell lines

• What is Cell Culture Media?

• Questions to help you choose the right cell culture medium

• Types of cell culture media

• Most importantly…

• References

Cell culture refers to the process of cultivating cells in a controlled, artificial environment for scientific research purposes. One of the first instances of cell culture was in 1907, when Ross Harrison was able to grow embryonic frog nerve fibers outside the body [1]. Today, cell culture has become an essential tool in laboratories worldwide, enabling rapid scientific progress in IVF (in vitro fertilization) technology, stem cell and cancer research, regenerative medicine, cell-based compound screens, and the production of biopharmaceuticals/pharmaceutical compounds like vaccines and therapeutic proteins [2]. Additionally, cell culture is playing a growing role in the food industry for testing contaminants and cellular agriculture, including cultured meat production.

Over the past few decades, a wide range of different types of cell cultures have been established, from various mammalian and insect species. The main types of cell lines that are used in cell cultures include:

• Primary cell lines: these are isolated directly from various mammalian tissues. Examples include epithelial cells, fibroblasts, and hematopoietic cells. (Learn more about what is primary cell culture )
  
  

• Continuous cell lines: these can proliferate indefinitely and can be cultured from genetically modified primary cell lines. Examples include the human cervical cancer cell line HeLa, the Chinese hamster ovary cell line CHO, the mouse Sertoli cell line MSC-1, and the butterfly ovary cell line Sf9.
  
  

• Stem cell lines: these are cultures of identical stem cells that can be differentiated into different cell types. Examples include pluripotent stem cell lines, such as human embryonic stem cell lines, and induced pluripotent stem cell lines, which can differentiate into various cell types. ( More about stem cells )
  
  

In general, primary cell lines are more sensitive and have more complex nutritional needs than continuous cell lines, requiring a specific set of nutrients, tissue-specific cytokines, and growth factors. Also, compared to primary and continuous cell lines, stem cell lines have unique metabolism and nutritional requirements that are needed to support their proliferation, and prevent stem cell differentiation, or senescence.

To meet the nutritional and environmental needs of your cell line, you need the right cell culture media. In this article, we discuss what cell culture media is and how to optimize your media choice to support your cell line and application.

Media provides cell cultures with nutrients, in addition to several other components that provide an optimal environment for cell growth, survival, and cellular function. This means that the quality of the media and the cellular environment that it provides are important considerations for your cell culture.

All basic media require the following components to support mammalian cell growth and meet the cells’ metabolic needs:

• 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: are needed to regulate membrane potential and osmolality.
  
  

• Amino acids: are the building blocks of proteins. Both essential and nonessential amino acids may be used to boost cell viability and growth.
  
  

• Serum: contains growth factors and inhibitors, hormones, amino acids, carbohydrates, lipids, vitamins, trace elements, minerals, and more that are needed for cellular growth. Serum from fetal and calf bovine sources are commonly used to support the growth of cells in culture. However, serum-free media are also commonly used in cell cultures.
  
  

• 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.
  
  

• Fatty acids and lipids: are particularly important in serum-free media as they are generally present in serum. Lipids and fatty acids are important as structural components of membranes, as a source of energy, and in cell signaling, transport, and biosynthesis.
  
  

• Proteins and peptides: are particularly important in serum-free media, which often has no or low protein content. Albumin, transferrin, and fibronectin are commonly used proteins in cell culture media, which support a range of cellular functions, including the transport of ligands, iron, and cellular attachment.
  
  

• Buffering systems: are important for the regulation of pH, such as HEPES or bicarbonate/CO2 systems. Serum can also increase the buffering capacity of cultures.
  
  

• Trace Elements: such as iron, potassium, magnesium, and zinc are necessary for cells to grow. For example, sources of elemental iron may contain trace quantities of manganese, an element that is essential for the glycosylation of proteins by animal cells.
  
  

• Antibiotics: are an optional addition to cell culture media, to inhibit fungal and bacterial growth which could otherwise contaminate cell cultures. However, the use of antibiotics in cell culture media can be toxic to specific cell lines, slowing cell growth and proliferation, and negatively affecting cellular metabolism.
  
  

Selecting the optimal media for your cell culture is a critical decision that depends on the specific cells being grown and the research objectives (Figure 2). This process can often be daunting, but by asking yourself the following questions, you can find the most suitable choice:

1. What is the best cell line to use for your research objective?
2. Are you considering the use of serum in your culture?
3. What specific nutritional requirements does your cell line have?
4. Are antibiotics suitable for your cell culture?

1. What is the best cell line to use for your research objective?

Before selecting your cell culture media, you should consider which cell line is best suited for the purpose of your experiment. For instance, when manufacturing biotherapeutic products such as antibodies, synthetic hormones, or enzymes, continuous cell cultures may be preferred, due to their longevity and lower nutritional requirements. However, for other applications, such as cellular disease models, specific primary cell lines, or stem cell lines may be the best choice.

In addition, it will be important to consider the vessel that you’re using to grow your cells, from flask to bioreactor. Whether you’re scaling up or wanting more control, bioreactors have very different feeding and mixing mechanisms from shaking flasks that will affect nutrient, pH, temperature, and oxygen requirements. Make sure to adjust your culture media choice for these conditions!

2. Are you considering the use of serum in your cell culture?

Another factor to consider is whether you would prefer to use serum in your culture or opt for serum-free alternatives. Classical media commonly consists of a basal media supplemented with animal serum as a source of nutrients e.g., MEM or DMEM. There are several advantages and disadvantages of using serum in cell culture media (Table 1).

While classical media that contains serum is easier to design and can be effective for a range of cell types, the use of animal sera has several disadvantages. Serum often contains ill-defined and variable components such as hydrolysates, growth factors, hormones, carrier proteins, and attachment factors. This means that serum can be unsuitable for studies that require reproducibility, and consistent performance, growth, or productivity. Animal serum can also be a source of bacterial contamination and therefore isn’t suitable for valuable or largescale cultures.

Several types of cell culture media have been developed that avoid the use of animal serum. This includes chemically defined media, which has known concentrations of all its chemical components, and contains no yeast, animal, or plant tissue. This improves the quality of the media, by reducing the risk of contamination and providing a consistent cellular environment between cultures and ensures that your results are reproducible.

It’s important to note that due to the higher cost, variability, and increasing regulatory requirements, research groups and biomanufacturing companies are increasingly switching from serum-based products to chemically defined, serum-free media.

3. What specific nutritional requirements does your cell line have?

Different cell types have varied nutritional requirements to support cell growth and maintenance, including a specific set of amino acids, vitamins, fatty acids, and lipids. For example, murine myeloma NS0 cells require exogenous cholesterol for optimal growth, while other mammalian cells that are commonly used in the biotechnology industry, like CHO and HEK293 cells, do not [3]. By thoroughly researching the cell line you’re planning to use, you will inevitably identify specific nutritional requirements needed for that cell line [4]. Understanding these requirements is essential to helping you choose the right cell media and identify any supplements that are needed to support your cultures.

4. Are antibiotics suitable for your cell line?

Another component of cell culture media that should be carefully considered is the addition of antibiotics. Bactericidal antibiotics are routinely used in cell culture media to prevent the spread of bacterial contamination. Despite their benefits, bactericidal antibiotics can have toxic effects on mammalian cell lines. For example, penicillin, streptomycin, and gentamicin can inhibit cell morphology, growth, proliferation, and differentiation. Bactericidal antibiotics can also cause severe metabolic and genetic defects in various primary, continuous, and stem cell lines [5-9].

Therefore, when considering the use of antibiotics, the specific antibiotic and its dosage should be investigated to ensure that your cell line isn’t negatively impacted. Alternatively, the use of antibiotics in cell culture media can be avoided altogether with the adoption of the correct aseptic techniques.

Once you’ve chosen the best cell line for your application and have identified the components that are required to provide an optimal cellular environment, the next step is to select the most appropriate type of cell culture media. Over the last 60 years, various defined media types have been developed, that are suitable for supporting the growth of a wide range of mammalian cell types and applications (Table 2):

When choosing the right type of cell media, it is crucial to understand the nutritional and environmental needs of your specific cell type, and whether additional factors such as antibiotics, serum, and supplements are required. But it doesn’t have to be perfect straight away! With some experimentation, you can ensure that your chosen media and any additional components are perfectly optimized for your particular project and cell line.

1. Harrison, R. G. (1910). The outgrowth of the nerve fiber as a mode of protoplasmic movement. Journal of Experimental Zoology, 9(4), 787–846. https://doi.org/10.1002/jez.1400090405

2. Jedrzejczak-Silicka, M. (2017). History of Cell Culture. In New Insights into Cell Culture Technology. IntechOpen. https://doi.org/10.5772/66905

3. Yao, T., & Asayama, Y. (2017). Animal-cell culture media: History, characteristics, and current issues. Reproductive Medicine and Biology, 16(2), 99. https://doi.org/10.1002/rmb2.12024

4. Sato, J. D., & Kan, M. (1998). Media for Culture of Mammalian Cells. Current Protocols in Cell Biology, 00(1), 1.2.1-1.2.15. https://doi.org/10.1002/0471143030.cb0102s00

5. Chang, Y., Goldberg, V. M., & Caplan, A. I. (2006). Toxic effects of gentamicin on marrow-derived human mesenchymal stem cells. Clinical orthopaedics and related research, 452, 242-9. https://doi.org/10.1097/01.blo.0000229324.75911.c7

6. Cohen, S., Samadikuchaksaraei, A., Polak, J. M., & Bishop, A. E. (2006). Antibiotics reduce the growth rate and differentiation of embryonic stem cell cultures. Tissue engineering, 12(7), 2025-30. https://doi.org/10.1089/ten.2006.12.2025

7. Llobet, L., Montoya, J., López-Gallardo, E., & Ruiz-Pesini, E. (2015). Side Effects of Culture Media Antibiotics on Cell Differentiation. Tissue engineering. Part C, Methods, 21(11), 1143-7. https://doi.org/10.1089/ten.TEC.2015.0062

8. Relier, S., Yazdani, L., Ayad, O., Choquet, A., Bourgaux, J.-F., Prudhomme, M., . . . David, A. (2016). Antibiotics inhibit sphere-forming ability in suspension culture. Cancer ell international, 16, 6. https://doi.org/10.1186/s12935-016-0277-6

9. Ryu, A. H., Eckalbar, W. L., Kreimer, A., Yosef, N., & Ahituv, N. (2017). Use antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced changes in gene expression and regulation. Scientific reports, 7(1), 7533. https://doi.org/10.1038/s41598-017-07757-w