The Cell Environment (including types of culture medium)

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 thirty 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 (Prod. No. T2397) was designed for use in work with primary mammalian cells. These have since been modified and enriched with amino acids, vitamins, fatty acids and lipids. Consequently media suitable for supporting the growth of a wide range of cell types are now available. 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 in the table below (Table 2).

Table 1: Different types of culture medium and their uses

Media type                      Examples                   Uses

                           PBS, Hanks BSS, Earles salts

Balanced salt solutions    DPBS (Prod. No. D8537 / D8662)  Form the basis of many complex media
 
                          HBSS (Prod. No. H9269 / H9394)

                           EBSS (Prod. No. E2888)

Basal media                MEM (Prod. No. M2279)           Primary and diploid cultures

                           DMEM (Prod. No. D5671)         Modification of MEM containing increased level of amino acids and vitamins. Supports a wide range of cell types including hybridomas

                           GMEM (Prod. No. G5154)         Glasgows modified MEM was defined for BHK-21 cells

Complex media              RPMI 1640 (Prod. No. R0883)     Originally derived for human leukaemic cells. It supports a wide range of mammalian cells including hybridomas

                           Iscoves DMEM (Prod. No. 13390)  Further enriched modification of DMEM which supports high density growth

                         Leibovitz L-15(Prod. No. L5520, liquid) Designed for CO2 free environments

                          TC 100 (Prod. No. T3160)

                         Grace's Insect Medium

                           (Prod. No. G8142)

                      Schneider's Insect Medium (Prod. No. S0146) Designed for culturing insect cells

Serum Free Media          CHO (Prod. No. C5467)             For use in serum free applications.
 
                       Ham F10 and derivatives

                       Ham F12 (Prod. No. N4888)

                     DMEM/F12 (Prod. No. D8062)           NOTE: These media must be supplemented with other factors such as insulin, transferrin and epidermal growth factor. These media are usually HEPES buffered

Insect cells                 Sf-900 II SFM,

                 SF Insect-Medium-2 (Prod. No. S3902)     Specifically designed for use with Sf9 insect cells 

1.Basic Constituents of media

Inorganic salts

Carbohydrates

Amino Acids

Vitamins

Fatty acids and lipids

Proteins and peptides

Serum

Each type of constituent performs a specific function as outlined below:

2.Inorganic Salts

The inclusion of inorganic salts in media performs several functions. Primarily they help to retain 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.

3.Buffering Systems

Most cells require pH conditions in the range 7.2 - 7.4 and close control of pH 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 acid conditions pH (7.0 - 7.4). 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 (Prod. No. H4034).

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 (Prod. No. H4034) 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. HEPES (Prod. No. H4034) buffered cultures do not require a controlled gaseous atmosphere.

Most commercial culture media include phenol red (Prod. No. P3532 / P0290) 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 (acid) or purple (alkali).

4.Carbohydrates

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 1g/l to 4.5g/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.

5.Vitamins

Serum is an important source of vitamins in cell culture. However, many media are also enriched with vitamins making 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 and 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.

6.Proteins and Peptides

These 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.

7.Fatty Acids and Lipids

Like proteins and peptides these are important in serum free media since they are normally present in serum. e.g. cholesterol and steroids essential for specialized cells.

8.Trace Elements

These include trace elements such as zinc, copper, selenium and tricarboxylic acid intermediates. Selenium is a detoxifier and helps remove oxygen free radicals.

Whilst all media may be made from the basic ingredients this is time consuming and may predispose to contamination. For convenience most media are available as ready mixed powders or as 10x and 1x liquid media. All commonly used media are listed in the Sigma-Aldrich Life Science Catalogue. If powder or 10x media are purchased it is essential that the water used to reconstitute the powder or dilute the concentrated liquid is free from mineral, organic and microbial contaminants. It must also be pyrogen free (Prod. No. W3500, water, tissue culture grade). In most cases water prepared by reverse osmosis and resin cartridge purification with a final resistance of 16-18Mx is suitable. Once prepared the media should be filter sterilized before use. Obviously purchasing 1x liquid media direct from Sigma eliminates the need for this.

9.Serum

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. Other types of serum are available including newborn calf serum and horse serum. The quality, type and concentration of serum can all 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.

Serum is also able to increase the buffering capacity of cultures that 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 whilst using a cell scraper. A further advantage of serum is the wide range 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-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 since 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 is screened for the presence of bovine viral diarrhoea virus (BVDV) and mycoplasma.
Heat inactivation of serum (incubation at 56oC for 30 minutes) can help to reduce the risk of contamination since some viruses are inactivated by this process. However the routine use of heat inactivated serum is not an absolute requirement for cell culture. The use of serum also has a cost implication not only in terms of medium formulation but also in downstream processing. A 10% FBS supplement contributes 4.8mg of protein per milliliter of culture fluid, which complicates downstream processing procedures.

10.Guidelines for serum use

Fetal bovine serum (FBS) has been used to prepare a number of biological and has an excellent record of safety. The recognition of Bovine spongiform encepalopathy (BSE) in 1986 and it’s subsequent spread into continental Europe along side the announcement of the probable link between BSE and a new variant of Creutzfeldt Jacob 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. As far as BSE is concerned, the EU guidelines on minimizing the risk of BSE transmission via medicinal products, CPMP/BWP/877/96, recommends the main measures to be implemented in o
rder to establish the safety of bovine material versus the BSE risk. Again, similarly the focus is on geographical origin, the age of the animals, the breeding and slaughtering conditions, the tissue to be used and the conditions of it’s 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.

Recent 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. Sigma-Aldrich has recognized this growing trend and works closely with customers to optimize animal free media formulations to meet each customer’s cell culture requirements.

Similarly the FDA has similar guidelines when accepting regulatory submissions. The FDA regulates all medicinal products for Human use, such as therapeutics, vaccines and diagnostics, and, usually, the United States Department Agriculture (USDA) are not involved.

The USDA regulates all medicinal products for veterinary use or for agricultural use. Similarly, the USDA regulates all products that contain a primary component of animal origin. It is important that you are aware of the use and restrictions when using serum, for further information contact you local Sigma-Aldrich sales office for specific information on the serum being used (contact information is at the back of this handbook). 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 be allowed into the USA.

11.Origin of Serum

USA/Canada, New Zealand, Finland and Denmark -No safety testing required. Australia, Mexico, Central America - Safety testing may be required, depending on the geographical region where the serum was collected.

Sigma-Aldrich carries out all safety test requirements stipulated by the USDA in USA laboratories. If necessary Sigma-Aldrich will assist customers in obtaining approval from the USDA, on any batch of serum of Australian origin supplied by Sigma-Aldrich.

Sigma-Aldrich sources serum from the states of Victoria and Tasmania. Tasmania will shortly be considered a separate geographical area within Australia eliminating the requirement for any safety testing.