Complete Growth Media

Animal Cell Culture Guide Complete Growth Media

Table of Contents

Cell Culture Media

Cell culture media are complex mixtures of salts, carbohydrates, vitamins, amino acids, metabolic precursors, growth factors, hormones, and trace elements. The requirements for these components vary among cell lines, and these differences are partly responsible for the extensive number of medium formulations. Carbohydrates are supplied primarily in the form of glucose. In some instances, glucose is replaced with galactose to decrease lactic acid build-up, as galactose is metabolized at a slower rate. Other carbon sources include amino acids (particularly L-glutamine) and pyruvate.

In addition to nutrients, the medium helps maintain the pH and osmolality in a culture system. The pH is maintained by one or more buffering systems; CO2/sodium bicarbonate, phosphate, and HEPES are the most common. Sera will also buffer a complete medium. Phenol red, a pH indicator, is added to medium to colorimetrically monitor changes in pH. Commonly used culture media include the following: 

Eagle’s Minimum Essential Medium (EMEM) was among the first widely used media and was formulated by Harry Eagle from his earlier and simpler basal medium (BME). BME was developed for culturing mouse L cells (ATCC® CCL-1™) and HeLa cells (ATCC® CCL-2™). Over time, there have been numerous variations on the EMEM formula for different applications. ATCC EMEM (ATCC® 30-2003™) contains Earle’s balanced salt solution, nonessential amino acids, and sodium pyruvate. It is formulated with a reduced sodium bicarbonate concentration (1,500 mg/L) for use with 5% CO2 (see Sodium Bicarbonate and Buffering in the Media Ingredients section below). Because EMEM is a simple medium, it is often fortified with additional supplements or higher levels of serum. 

Dulbecco’s Modified Eagle’s Medium (DMEM) has roughly twice the concentration of amino acids and four times the amount of vitamins as EMEM, as well as ferric nitrate, sodium pyruvate, and some supplementary amino acids (though not all nonessential amino acids). The original formulation contained 1,000 mg/L of glucose, but in the more commonly used variations this amount was increased to 4,500 mg/L. 

ATCC DMEM (ATCC® 30-2002™) has 4,500 mg/L of glucose and a reduced sodium bicarbonate concentration (1,500 mg/L) for use with 5% CO2

Iscove’s Modified Dulbecco’s Medium (IMDM) was formulated for growth of lymphocytes and hybridomas. Compared to DMEM, it has additional amino acids, vitamins and inorganic salts. Potassium nitrate was substituted for ferric nitrate. It also contains HEPES and selenium. ATCC IMDM (ATCC® 30-2005™) has a reduced sodium bicarbonate concentration (1,500 mg/L) for use with 5% CO2

Hybri-Care Medium (ATCC® 46-X™) is a combination and modification of DMEM and NCTC 135 medium supplemented with insulin, oxalacetic acid, and HEPES. It is based on the formulation used by David H. Sachs and collaborators5 for the propagation of hybridomas and other fastidious cell lines. 

McCoy’s 5A and RPMI-1640 were developed at Roswell Park Memorial Institute (RPMI) in Buffalo, New York. McCoy’s 5A (ATCC® 30-2007™) was originally used to grow Novikoff hepatoma cells and will support the growth of primary cultures. 

RPMI-1640 is a modification of McCoy’s 5A and was developed for the long-term culture of peripheral blood lymphocytes. RPMI-1640 will support the growth of a wide variety of cells in suspension as well as a number of cells grown as monolayers.

ATCC RPMI-1640 (ATCC® 30-2001™) was modified to contain higher amounts of glucose (4,500 mg/L), sodium pyruvate, and HEPES buffer. It also contains a reduced concentration of sodium bicarbonate (1,500 mg/L) for use with 5% CO2

Ham’s Nutrient Mixtures were originally developed to support the clonal outgrowth of Chinese hamster ovary (CHO) cells (ATCC® CCL-61™). As with EMEM, there have been numerous modifications to the original formulation including Ham’s F-12 medium, a more complex formulation than the original F-10 suitable for serum-free propagation. 

Kaighn’s modification of Ham’s F-12 (Ham’s F-12K) was designed to support the growth and differentiation of primary cells with or without serum. F-12K has increased amounts of amino acids, pyruvate, biotin, calcium, magnesium, putrescine, and phenol red in addition to other modifications from the F-12 formula. ATCC Ham’s F-12K (ATCC® 30-2004™) has a reduced sodium bicarbonate concentration (1,500 mg/L) for use with 5% CO₂. 

DMEM/F12 Medium is a 1:1 mixture of Dulbecco’s modified EMEM and Ham’s F-12. It is an extremely rich and complex medium and will support the growth of a broad range of cell types in both serum and serumfree formulations. 

ATCC DMEM/F12 medium (ATCC® 30-2006™) has a reduced sodium bicarbonate concentration (1,500 mg/L) for use with 5% CO2

Leibovitz’s L-15 Medium (ATCC® 30-2008™) is formulated for use without CO2 incubation as is found in teaching laboratories or when collecting biopsy samples. The standard sodium bicarbonate/CO2 buffering system is replaced by a combination of phosphate buffers, free-base amino acids, higher levels of sodium pyruvate, and galactose. Cell cultures can be grown in CO2 incubators with L-15 medium provided there is no exchange between the air in the culture vessel with that of the incubator (i.e., caps of flasks are tightly closed).

Media Formulations

Formulations of media available from ATCC can be found online. Please note that there are cell lines in the collection that require media not currently sold by ATCC.

Media Ingredients

Sodium bicarbonate and buffering Cells produce and require small amounts of carbon dioxide for growth and survival.6 In culture media, dissolved CO2 is in equilibrium with bicarbonate ions and many medium formulations take advantage of this CO2/bicarbonate reaction to buffer the pH of the medium. CO2 dissolves freely into the medium and reacts with water to form carbonic acid. As the cells metabolize and produce more CO2, the pH of the medium decreases as the chemical reaction below is driven to the right:

H2O + CO2 <—> H2CO3 <> H+ + HCO3-

The optimal pH range of 7.2 to 7.4 can be maintained by supplementing the medium with sodium bicarbonate and regulating the level of CO₂ in the atmosphere above the medium as shown by the reaction below: 

H2O + CO2 + NaHCO<>  H+ + Na+ + 2HCO3-

In tissue culture, cells are grown either in open systems (where there is free exchange of the atmosphere immediately above the medium with the atmosphere of the incubator) or in closed systems (where the two atmospheres are kept separate). The buffering system employed in the medium needs to be matched to the culture system. Otherwise the cells may be subject to metabolic stress which will impair their performance.

In closed systems the level of CO2 is regulated by the metabolism of the cells. The culture vessel must be sealed (flasks tightly capped) to retain any CO2 generated by the cells. Consequently, closed systems provide additional protection against contamination and have simpler incubator requirements than open systems. Closed systems usually require media with buffers based on Hanks’ balanced salt solution having relatively low levels of sodium bicarbonate. 

In open systems, humidity (to reduce evaporation) and a means of regulating CO2 levels (if the culture medium contains sodium bicarbonate) are required during incubation to maintain the pH of the culture medium. Open systems usually require the higher levels of sodium bicarbonate found in Earle’s salt solution combined with a 5 to 10% CO2 atmosphere supplied by the incubator. In general, 1.2 g/L to 2.2 g/L of sodium bicarbonate is used with 5% CO2 whereas 3.7 g/L sodium bicarbonate is used with 10% CO2. The exact amount will depend upon the medium formulation. 

In some cases, researchers “gas” the atmosphere of the culture vessel with a stream of sterile-filtered 5% CO2/95% air mixture and then tightly seal the flask prior to incubation in a nonhumidified and non-CO2 incubator.7 While these culture vessels work with simpler non-humidified, non-CO2 incubators, the medium requirements are those of an open system. 

All ATCC media, with the exception of Leibovitz’s L-15 (ATCC® 30-2008™), are designed to be used with 5% CO2 levels. Most have a sodium bicarbonate concentration of 1.5 g/L and are supplemented with extra sodium pyruvate. ATCC modification of McCoy’s 5A (ATCC® 30-2007™) has a slightly higher levels of sodium bicarbonate (2.2 g/L) and does not contain sodium pyruvate. 

While most commercial formulations of liquid media do contain the appropriate amount of sodium bicarbonate, it is generally omitted from the powdered form and needs to be added before use. Some medium formulations incorporate other buffering systems such as phosphate or HEPES in addition to CO2/ sodium bicarbonate. These media have the advantage of maintaining optimal pH in an open system when the culture vessel is removed from the enriched CO2 atmosphere of the incubator.

HEPES buffer 

HEPES and other organic buffers can be used with many cell lines to effectively buffer the pH of the medium.8 Indeed, some standard medium formulations include HEPES. However, this compound can be toxic, especially for some differentiated cell types, so evaluate its effects before use.9 HEPES has been shown to greatly increase the sensitivity of media to the phototoxic effects induced by exposure to fluorescent light.10,11 

Phenol red 

Phenol red is used to monitor the pH of media. During cell growth, the medium changes color as it changes pH due to metabolites released by the cells. At low pH levels, phenol red turns the medium yellow, while at higher pH levels it turns the medium purple. For most tissue culture work (pH 7.4), the medium should be bright red. 

Unfortunately, phenol red can mimic the action of some steroid hormones, particularly estrogen. For studies with estrogen-sensitive cells, such as mammary tissue, use media without phenol red. Additionally, the sodium-potassium ion homeostasis is upset when phenol red is included in some serum-free formulations; this effect is neutralized by the inclusion of serum or bovine pituitary hormone in the medium.12 Phenol red is frequently omitted from studies with flow cytometry as its color interferes with detection.

L-Glutamine 

L-Glutamine (ATCC® 30-2214™) is an essential amino acid required by virtually all mammalian and insect cells grown in culture. It is used for protein production, as an energy source, and in nucleic acid metabolism. It is also more labile in liquid cell culture media than other amino acids. The rate and extent of L-glutamine degradation are related to storage temperatures, age of the product, and pH. 

Because L-glutamine is so labile, it is often omitted from commercial liquid medium preparations to lengthen the product shelf life. In these cases, it must be aseptically added prior to use. L-Glutamine is not as labile in dry form and most powdered medium formulations do include it. 

In some cases, the addition of L-glutamine to complete cell culture medium can extend the usable life of the medium. If L-glutamine is suspected to be a limiting factor during cell culture, a simple test of ‘spiking’ the medium with a small amount of L-glutamine will determine whether or not more is required. Simply add a small amount of L-glutamine (~2 mM final concentration) to the culture medium. If the cell growth rate increases, L-glutamine is most likely deficient and more should be added. Alternately, the concentration of L-glutamine can be measured directly by standard analytical means such as HPLC (High Performance Liquid Chromatography).

L-Glutamine concentrations for mammalian cell culture media can vary from 0.68 mM in Medium 199 to 4 mM in Dulbecco’s Modified Eagle’s Medium. Invertebrate cell culture media, such as Schneider’s Drosophila medium, may contain as much as 12.3 mM L-glutamine. 

Use caution when adding more L-glutamine than is called for in the original medium formulation. L-Glutamine degradation results in the build-up of ammonia which can have a deleterious effect on some cell lines. For most cell lines, ammonia toxicity is more critical for cell viability than L-glutamine limitation.

Nonessential amino acids

All medium formulations contain the ten essential amino acids as well as cysteine, glutamine, and tyrosine. The inclusion of the other non-essential amino acids (alanine, asparagine, aspartic acid, glycine, glutamic acid, proline, and serine) in some media formulations reduces the metabolic burden on the cells allowing for an increase in cellular proliferation. 

Sodium pyruvate 

Pyruvate is an intermediary organic acid metabolite in glycolysis and the first component of the Embden-Meyerhof pathway. It can pass readily into or out of the cell. Its addition to tissue culture medium provides both an energy source and a carbon skeleton for anabolic processes. Pyruvate may help in maintaining certain specialized cells, in clonal selection, in reducing the serum concentration of the medium,7 and in reducing fluorescent light-induced phototoxicity.10 Cellular metabolism of pyruvate produces carbon dioxide which is given off into the atmosphere and becomes bicarbonate in the medium. Sodium pyruvate is added to give a final concentration of 1 mM in most media, but is increased to 5 mM in Leibovitz’s L-15 medium primarily to facilitate use in CO2-free environments.


Media Supplements

The complete growth media recommended for some cell lines requires the addition of components not already available in the base media and serum. These components include hormones, growth factors and signaling substances that sustain proliferation and maintain normal cell metabolism.

Supplements are usually prepared as 100× (or higher) stock solutions in serum-free medium. Some supplements may need to be dissolved in a solvent prior to subsequent dilution in serum-free medium to the stock concentration. Stock concentrations should be aliquoted into small volumes and stored at an appropriate temperature; most stock concentrations can be stored at -80°C, but check with your supplier prior to storing. 

The addition of supplements can change the final osmolality of the complete growth medium, which may have a negative effect on the growth of cells in culture. It is best to recheck the osmolality of the complete growth medium after small volumes of supplement stock solutions are added; optimal osmolality for most vertebrate cell lines should fall between 260 mOSM/kg and 320 mOSM/kg. 

After supplements have been added to a base medium, the shelf life of the complete growth medium should be determined on a case-by-case basis. Complete media containing protein supplements (e.g., epidermal growth factor, bovine serum albumin) tend to degrade faster than base media alone. Most complete growth media can be stored in aliquots at 2°C to 8°C for about a month. However, if any supplement is expected to expire before the one-month period has passed, the expiration date for the complete growth media should follow suit. Some fastidious cell lines may require that components be added immediately before use. Do not freeze complete growth medium. Freezing cell culture media at -70°C or below causes some of the growth factors and/or vitamins to precipitate out of solution. It can be very difficult to get these components to go back into solution after thawing, even if warmed to 37°C. ATCC recommends storing media between 2°C and 8°C, away from light. 

For additional information regarding the preparation, storage, or usage of specific supplements, contact your local supplier or consult with the manufacturer’s Product Information Sheet.

Osmolality

The osmolality of cell culture media for most vertebrate cells is kept within a narrow range from 260 mOsm/kg to 320 mOsm/kg, even though most established cell lines will tolerate a rather large variation in osmotic pressure. In contrast, the osmolality requirements for some invertebrate cell lines fall outside of this range. For example, the snail embryo (ATCC® CRL-1494™) requires medium of about 155 mOsm/kg, while some insect cells prefer 360 mOsm/kg to 375 mOsm/kg. Most commercially available liquid media report osmolality and it is advisable to check the osmolality of any medium after the addition of saline solutions, drugs or hormones dissolved in an acid or base solution, or large volumes of buffers (e.g., HEPES).

Antibiotics and Antimycotics

Antibiotics and/or antimycotic agents are added to cell culture media as a prophylactic to prevent contamination, as a cure once contamination is found, to induce the expression of recombinant proteins, or to maintain selective pressure on transfected cells.

Routine use of antibiotics or antimycotics for cell culture is not recommended unless they are specifically required, such as G418 for maintaining selective pressure on transfected cells. Antibiotics can mask contamination by mycoplasma and resistant bacteria. Further, they can interfere with the metabolism of sensitive cells. Avoid antimycotics as they can be toxic to many cell lines. 

While cell lines can be cured of microbial contamination with antibiotics and/or antimycotics, this is not recommend unless the cell line is irreplaceable; the process is lengthy and there is no guarantee contamination will be eliminated. Even if the contamination is eliminated, there is no way of ensuring that the resulting cell line will have the same characteristics as the initial one due to the stress of the treatment. It is best to discard the cell line and start over with new stocks. Mycoplasma contamination in particular is very difficult to eliminate (See section on Mycoplasma Contamination). In some cases, antibiotic use for short periods of time can serve as a valuable prophylactic. For example, antibiotic use is recommended when developing and working with primary culture and when using flow cytometry to isolate subpopulations.

If an antibiotic is used in medium, penicillin-streptomycin solution (ATCC® 30-2300™) can be added at 0.5 to 1 mL of solution per 100 mL of cell culture medium for a final concentration of 50 to 100 IU/mL penicillin and 50 to 100 µg/mL streptomycin. Gentamicin sulfate, another antibiotic (ATCC® 30-2303™), is used at 50 to 100 µg/mL. The antimycotic amphotericin B (ATCC® 30-2301™) is used at 2.5 µg/mL.13 These concentrations apply to media that contain serum. For serum-free media, reduce the concentrations by at least 50%.

Animal Sera

Sera serve as a source for amino acids, proteins, vitamins (particularly fat-soluble vitamins such as A, D, E, and K), carbohydrates, lipids, hormones, growth factors, minerals, and trace elements. Additionally, serum buffers the culture medium, inactivates proteolytic enzymes, increases medium viscosity (which reduces shear stress during pipetting or stirring), and conditions the growth surface of the culture vessel. The exact composition is unknown and varies from lot to lot, although lot-to-lot consistency has improved in recent years.

Sera from fetal and calf bovine sources are commonly used to support the growth of cells in culture. Fetal serum is a rich source of growth factors and is appropriate for cell cloning and for the growth of fastidious cells. Calf serum, because of its lower growth-promoting properties, is used in contact-inhibition studies with NIH/3T3 cells (ATCC® CRL-1658™). In contrast to fetal or calf sera, horse serum is collected from a closed herd of adult animals ensuring lot-to-lot consistency. Horse serum is less likely to carry the contaminants found in bovine sera such as viruses and less likely to metabolize polyamines which may be mitogenic for some cells. Horse and bovine calf sera are less expensive and more readily available than fetal bovine serum. The pricing and availability of fetal serum fluctuates considerably. 

Unfortunately, naturally derived products from bovine sources may contain adventitious viruses such as bovine viral diarrhea virus (BVDV), bovine parvovirus, bovine adenovirus, and blue tongue virus. All reputable suppliers test their products for infectious virus by several methods including fluorescent antibody, cytopathic effect, and hemadsorption. These products are also screened for the standard microbial contaminants such as bacteria, fungi, and mycoplasma. 

BVDV, in contrast to the other virus contaminants, is present in nearly all bovine serum at very low levels even when tests for infectious virus are negative. Fortunately, very few cell lines (except those of bovine origin) are susceptible to this virus. For the few sensitive cell lines, use non-bovine sera or irradiated bovine sera. Several ATCC cell lines were tested for BVDV contamination14 and the results of this study are indicated in the cell line description on the website. Bovine-derived products also may contain the agent responsible for bovine spongiform encephalopathy (BSE). Unfortunately, there is no test for the presence of this agent and we highly recommend that you obtain all bovine products (including sera) from countries not affected by BSE such as the United States, Australia and New Zealand. 

At one time animal serum was a major source of mycoplasma contamination of tissue culture cells. However, nearly all sera today are filtered through several 0.1-µm pore (or smaller) filters which effectively remove this organism.

ATCC offers the following four types of animal sera: 

  • Fetal Bovine Serum (also known as fetal calf) — ATCC® 30-2020
  • Fetal Bovine Serum qualified for embryonic stem cells — ATCC® SCRR-30-2020
  • Iron-supplemented Calf Bovine Serum — ATCC® 30-2030
  • Horse Serum — ATCC® 30-2040

These products are rigorously tested for adventitious infective agents and sourced from only U.S. herds. Further, each lot is tested for its ability to support cell growth and is the same sera used in ATCC labs.

Storage 

Store sera at -20°C or colder for storage over 30 days. ATCC sera are routinely stored at -70°C. Do not store sera at temperatures above -20°C for any length of time. Avoid repeated freeze-thaws by dispensing and storing in aliquots.

Thawing 

The following procedure is used to thaw serum: 

  1. Place frozen serum in a refrigerator at 2°C to 8°C overnight. 
  2. Put the bottles in a 37°C water bath and gently agitate from time to time to mix the solutes that tend to concentrate at the bottom of the bottle. 

Do not keep the serum at 37°C any longer than necessary to thaw it, and do not thaw the serum at higher temperatures. Thawing serum in a bath above 40°C without mixing may lead to the formation of a precipitate inside the bottle.

Turbidity and precipitates 

All sera may retain some fibrinogen. Because external factors may initiate the conversion of fibrinogen to fibrin, flocculent material or turbidity may be observed after serum is thawed. The presence of this material does not alter the serum’s performance. If the presence of flocculent material or turbidity is a concern, it can be removed by filtration through a 0.45-µm filter. 

A precipitate can form in serum when incubated at 37°C or higher for prolonged periods of time which may be mistaken for microbial contamination. This precipitate may include crystals of calcium phosphate, but does not alter the performance of the serum as a supplement for cell culture. Heat inactivation of sera can also cause the formation of precipitates.

Heat inactivation 

ATCC does not routinely use heat-inactivated serum unless specifically required for a particular cell line. Heat inactivation is usually unnecessary and can be detrimental to the growth of some cells. It will reduce or destroy growth factors present in the serum. 

Heat inactivation was originally performed to inactivate complement (a group of proteins present in sera that are part of the immune response) as well as to destroy mycoplasma contaminants. Today, mycoplasma contamination, if any, is removed by filtration. Removal of complement is usually unnecessary, but can be important when preparing or assaying viruses or in cytotoxicity tests. According to a study by HyClone,15 warming serum to 37°C inactivates heat-labile complement factors. A few types of cell lines grow better in heat-inactivated sera such as embryonic stem cells16 and many insect cell lines.17

The following procedure can be used to heat-inactivate serum: 

  1. Thaw serum. 
  2. Preheat a water bath to 56°C. Use sufficient water to immerse the bottle above the level of serum. 
  3. Mix thawed serum by gentle inversion and place in the 56°C bath. The temperature of the water bath will drop. 
  4. When the temperature of the water bath reaches 56°C again, continue to heat for an additional 30 minutes. Mix gently every 5 minutes to insure uniform heating. 
  5. Remove serum from water bath, cool quickly (slow cooling can sometimes reverse the inactivation of complement activity), and store at -20°C or colder.