Media and Supplements in Cell Culture

ECACC Laboratory Handbook 4th Edition

Cell Culture Media Types

In general terms, cultured cells require a sterile environment and a supply of nutrients for growth. In addition, the culture environment should be stable in terms of pH and temperature. Over the last 60 years, various defined basal media types have been developed and are now available commercially. Originally, balanced salt solutions were used to maintain contractility of mammalian heart tissue and Tyrode’s salt solution was designed for use in work with primary mammalian cells. These founding formulations have since been modified and enriched with amino acids, vitamins, fatty acids and lipids so that modern culture media are suitable for supporting the growth of a wide range of cell types. The precise media formulations have often been derived by optimizing the concentrations of every constituent. Examples of the different media and their uses are given below:

Table 1Different types of culture media and their uses

Basic Cell Culture Media Components


Serum is a complex mix of albumins, growth factors and growth inhibitors and is probably one of the most important components of cell culture medium. The most commonly used serum is fetal bovine serum (FBS). Other types of serum are available including newborn calf serum and horse serum. The quality, type and concentration of serum can affect the growth of cells and it is therefore important to screen batches of serum for their ability to support the growth of cells. In addition, there are other tests that may be used to aid the selection of a batch of serum including cloning efficiency, plating efficiency and the preservation of cell characteristics.

Fetal Bovine Serum

Figure 2.Fetal Bovine Serum

Serum can also increase the buffering capacity of cultures, which can be important for slow growing cells or where the seeding density is low (e.g. cell cloning experiments). It also helps to protect against mechanical damage which may occur in stirred cultures or when using a cell scraper.

A further advantage of serum is the wide range of cell types with which it can be used despite the varying requirements of different cultures in terms of growth factors. In addition, serum is able to bind and neutralize toxins. However, serum is subject to batch-to-batch variation that makes standardization of production protocols difficult.

There is also a risk of contamination associated with the use of serum. These risks can be minimized by obtaining serum from a reputable source, as suppliers of large quantities of serum perform a battery of quality control tests and supply a certificate of analysis with the serum. In particular, serum for cell culture must be screened for the presence of bovine viral diarrhea virus (BVDV) and mycoplasma. Heat inactivation of serum (incubation at 56 °C for 30 minutes) can help to reduce the risk of contamination, since some viruses are inactivated by this process; however, this process also denatures some proteins and destroys nutrients in the serum. With the advent of modern production methods for serum the routine use of heat inactivated serum is no longer a requirement for cell culture. The use of serum also has a cost impact, not only in terms of medium formulation but also in downstream processing. A 10% FBS supplement contributes 4.8 mg/mL of protein which complicates downstream processing procedures such as protein purification.

Guidelines for Serum Use

Fetal bovine serum (FBS) has been used to prepare a number of biologicals and has an excellent record of safety. The recognition of Bovine Spongiform Encephalopathy (BSE) in 1986 and its subsequent spread into continental Europe, alongside the announcement of the probable link between BSE and a new variant of Creutzfeldt-Jakob disease in humans, stimulated an increased concern about safe sourcing of all bovine materials. In 1993, the Food and Drug Administration (FDA) “recommended against the use of bovine derived materials from cattle which have resided in, or originated from countries where BSE has been diagnosed”.

The current European Union (EU) guidelines on viral safety focus on sourcing, testing and paying particular attention to the potential risk of cross contamination during slaughtering or collection of the starting tissue. Regarding BSE, the EU guidance on minimizing the risk of BSE transmission via medicinal products, EMEA/410/01 Rev. 3, recommends the principal measures to be implemented in order to establish the safety of bovine material. Again, the focus is on geographical origin, the age of the animals, breeding and slaughtering conditions, the tissue to be used and the conditions of its processing.

The use of FBS in production processes of medicinal products is acceptable provided good documentation on sourcing, age of the animals and testing for the absence of adventitious agents is submitted. All responsible suppliers of FBS for bio-pharmaceutical applications will provide such documentation.

Regulatory requirements in Europe stress the importance of justifying the use of material of bovine, caprine or ovine origin in the production of pharmaceutical products. Thus, although FBS has been used for many years in the production process of many medicinal products such as viral vaccines and recombinant DNA products, at present there is a justified trend to remove all material of animal origin from manufacturing processes. We have recognized this growing trend and works closely with customers to optimize animal free media formulations to meet each customer’s cell culture requirements. Serum-free cell lines that have been adapted to media that do not contain serum are available from ECACC.

The United States Department of Agriculture (USDA) regulates all products that contain a primary component of animal origin. With specific reference to serum the USDA has declared that for materials which fall under their jurisdiction, only biological products manufactured using serum from approved countries of origin will be allowed in to USA.

Origin of Serum

ECACC only uses serum sourced from countries with a negligible risk of BSE. Historically, serum sourced from Australia, New Zealand, and the USA has offered the lowest risk of BSE contamination. It is essential to check the source country of serum used and their BSE risk status. Use of serum of the appropriate quality is particularly important if the intended use of the serum is in the production of medicinal or other products being sent to the USA.

Serum from a reputable supplier should have undergone various quality control tests which will be listed in the product information sheet. Most serum products are cell culture tested for growth promotion, cloning efficiency and plating efficiency.

Standard tests performed on serum commonly include tests to determine the presence and/or level of the following: Sterility, Virus Contamination, Mycoplasma Contamination, Endotoxin, Hemoglobin, Total Protein, Immunoglobulin, Hormone Testing, pH (at room temperature) and Osmolality.

Inorganic Salts

The inclusion of inorganic salts in media performs several functions. Salts principally serve to establish and maintain the osmotic balance of the cells and help regulate membrane potential by provision of sodium, potassium and calcium ions. All of these are required in the cell matrix for cell attachment and as enzyme cofactors.

Buffering Systems

Most cells require pH conditions in the range 7.2-7.4 and close control of pH by a buffering system is essential for optimum culture conditions. There are major variations to this optimum, fibroblasts prefer a higher pH (7.4-7.7) whereas continuous transformed cell lines require more acidic conditions.

Regulation of pH is particularly important immediately following cell seeding when a new culture is establishing and is usually achieved by one of two buffering systems; (i) a “natural” buffering system where gaseous CO2 balances with the CO3/HCO3 content of the culture medium and (ii) chemical buffering using a zwitterion called HEPES.

Cultures using natural bicarbonate/CO2 buffering systems need to be maintained in an atmosphere of 5-10% CO2 in air usually supplied in a CO2 incubator. Bicarbonate/CO2 is low cost, non-toxic and also provides other chemical benefits to the cells.

HEPES has superior buffering capacity in the pH range 7.2-7.4 but is relatively expensive and can be toxic to some cell types at higher concentrations (above ~100 nM). HEPES buffered cultures do not require a controlled gaseous atmosphere.

Most commercial culture media include phenol red as a pH indicator so that the pH status of the medium is constantly indicated by the color. Usually the culture medium should be changed/replenished if the color turns yellow (acidic) or purple (basic).


The main source of energy is derived from carbohydrates generally in the form of sugars. The major sugars used are glucose and galactose; however, some media contain maltose or fructose. The concentration of sugar varies from basal media containing 1 g/L to 4.5 g/L in some more complex media. Media containing the higher concentration of sugars are able to support the growth of a wider range of cell types. Pyruvate is included in the formulation of some media, as an alternative energy source.

Amino Acids

Amino acids are the building blocks of proteins. ‘Essential’ amino acids must be added to culture media as cells are not able to synthesize these themselves. The concentration of amino acids in the culture medium will determine the maximum cell density that can be achieved - once depleted, the cells will no longer be able to proliferate.

In relation to cell culture, the essential amino acid glutamine is particularly significant. In liquid media or stock solutions glutamine degrades relatively rapidly. Optimal cell performance usually requires supplementation of the media with glutamine prior to use. Some media formulations include the more stable L-alanyl glutamine and do not require supplementation.

Adding supplemental non-essential amino acids to media both stimulates growth and prolongs the viability of the cells in culture.


Serum is an important source of vitamins in cell culture. Many media are also enriched with specific vitamins that make them consistently more suitable for a wider range of cell lines. Vitamins are precursors for numerous co-factors. Many vitamins, especially B group vitamins, are necessary for cell growth and proliferation, for some lines the presence of B12 is essential. Some media also have increased levels of vitamins A and E. The vitamins commonly used in media include riboflavin, thiamine and biotin.

Proteins and Peptides

Adequate proteins and peptides are particularly important in serum-free media. The most common proteins and peptides include albumin, transferrin, fibronectin and fetuin and are used to replace those normally present through the addition of serum to the medium.

Fatty Acids and Lipids

Like proteins and peptides, lipids and fatty acids are critical additives to serum-free media since they are normally present serum. Cholesterol and steroids are examples of macromolecules in this category that are essential for specialized cells.

Trace Elements

Trace elements essential for cultured cells include zinc, copper, selenium and tricarboxylic acid intermediates. Selenium is a detoxifier and helps remove oxygen free radicals.


Use of antibiotics in cell culture minimizes the loss of valuable cells, reagents, time and efforts due to contamination. Experienced researchers recommend cell culture-tested antibiotics, at adequate concentrations, be used while culturing cells. Pen-Strep is a mixture of penicillin and streptomycin widely used in mammalian cell culture media to prevent bacterial contamination. Certain antibiotics, such as puromycin, neomycin, and hygromycin, also function as selection agents, used to select and establish transfected/genetically modified cells for research purposes.

Preparation of Media

While any defined media may be made from its constituent ingredients it is time consuming for most labs performing cell culture, and may increase the risk of contamination. For convenience, many commonly used media are available as ready mixed powders or as 10x and 1x liquid media. All commonly used media are listed in our online catalogue. If powder or 10x media are used, it is essential that the water used to reconstitute the powder or dilute the concentrated liquid is tissue culture-grade and free from mineral, organic and microbial contaminants, including pyrogens. In most cases water prepared by reverse osmosis and resin cartridge purification with a final resistance of 16-18 MΩ is suitable. Once prepared the pH of the medium should be adjusted appropriately and then the media filter sterilized using a Stericup® filtration unit before use.