The Cell Environment (including types of culture medium)

Fundamental Techniques in Cell Culture Laboratory Handbook - 2nd Edition

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 30 years various defi ned 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 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 optimising the concentrations of every constituent. Examples of the different media and their uses are given in Table 2.


Table 2. Different types of culture medium and their uses (S3777)

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6.1 Basic Constituents of Media

  • Inorganic salts
  • Carbohydrates
  • Amino Acids
  • Vitamins
  • Fatty acids and lipids
  • Proteins and peptides
  • Serum
  • Trace Elements

Each type of constituent performs a specifi c function as outlined below:

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6.2 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.

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6.3 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.

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 benefi ts 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. 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 colour. Usually the culture medium should be changed/replenished if the colour turns yellow (acid) or purple (alkali).

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6.4 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.

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6.5 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, glutamine, an essential amino acid, is particularly signifi cant. In liquid media or stock solutions glutamine degrades relatively rapidly. Optimal cell performance usually requires supplementation of the media with glutamine prior to use.

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

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6.6 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 ribofl avin, thiamine and biotin.

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6.7 Proteins and Peptides

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

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6.8 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 specialised cells.

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6.9 Trace Elements

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

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6.10 Preparation of Media

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 and of tissue culture grade. In most cases water prepared by reverse osmosis and resin cartridge purifi cation with a fi nal resistance of 16-18MΩ is suitable. Once prepared, the media should be fi lter sterilised before use.

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6.11 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 foetal bovine serum (FBS). 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 effi ciency, plating effi ciency 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 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 neutralise toxins. However, serum is subject to batch-to-batch variation that makes standardisation of production protocols difficult.

There is also a risk of contamination associated with the use of serum. These risks can be minimised by obtaining serum from a reputable source since suppliers of large quantities of serum perform a battery of quality control tests and supply a certifi cate 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 56°C 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 millilitre of culture fl uid which complicates downstream processing procedures such as protein purification.

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6.12 Guidelines for Serum Use

Foetal 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 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 guidance on minimising the risk of BSE transmission via medicinal products, EMEA/410/01 Rev. 2, recommends the main measures to be implemented in order to establish the safety of bovine material. 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 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 justifi ed trend to remove all material of animal origin from manufacturing processes. Sigma- Aldrich has recognised this growing trend and works closely with customers to optimise 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 specifi c 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.

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6.13 Origin of Serum

ECACC only uses serum of Zone 1 origin, sterile fi ltered and cell culture tested. Zone 1 countries have BSE –free status such as the USA, Canada, Australia and New Zealand. It is essential to check the source country of the serum used and their Zone status. Sera from Mexico and Central American countries may require additional documentation to prove the geographical region of the donor herd to ensure BSE-free status. This is very 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 including growth promotion, cloning effi ciency and plating effi ciency tests.

Standard tests performed on serum commonly include tests to determine the presence and/or level of the following:

  • Sterility
  • Virus Contamination
  • Mycoplasma Contamination
  • Endotoxin
  • Haemoglobin
  • Total Protein
  • Immunoglobulin
  • Hormone Testing
  • pH (at room temperature)
  • Osmolality

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Did You Know?

  • In culture medium glutamine degrades rapidly to toxic ammonia at 37°C. Therefore aliquot your medium before use and only warm and use volumes you need that day. Discard opened bottles of prepared medium containing glutamine after 4-6 weeks.
  • The most commonly used serum is foetal bovine serum. Other types of serum are available including newborn calf serum and horse serum.

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Materials

     
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