Basic Constituents of media

Amino Acids

Amino acids are a required energy source for cells, influencing cell density, proliferation, and viability. A formulation providing the nonessential amino acids may minimize the metabolic burden of the cell to have to produce them, thus increasing cellular proliferation. Nonessential amino acids may also be added to the medium to replace those that have been depleted during growth.

Carbohydrates

Glucose is the most common carbohydrate used in mammalian cell culture and provides the major energy, or carbon source, for biosynthesis. Galactose may be substituted for glucose because it is metabolized at a slower rate, thereby decreasing any lactic acid build-up.

Fatty acids and lipids

These are also supplied by serum and are often needed by certain cell lines. For example, cholesterol – dependent hybridomas require cholesterol in the medium for growth.

Inorganic salts

Inorganic salts contribute to the basal media osmolality. Their presence in the medium, acts to regulate the membrane potential of the cells. Salts are also needed for cell attachment.

Proteins and peptides

Serum is a rich source of proteins. Some proteins found in serum are albumin, aprotinin, fetuin, fibronectin, and tranferrin. Albumin is main protein in blood acting to bind water, salts, free fatty acids, hormones, and vitamins and transport them between tissues and cells. The binding capacity of albumin makes it a suitable remover of toxic substances from the cell culture media. Aprotinin is a protective agent in cell culture systems, stable at neutral and acidic pH and resistant to high temperatures and degradation by proteolytic enzymes. It has the ability to inhibit several serine proteases such as trypsin. Fetuin is a glycoprotein found in fetal and newborn serum at larger concentrations than in adult serum. It is also an inhibitor of serine proteases. Fibronectin is a key player in cell attachment. Transferrin is an iron transport protein that acts to supply iron to the cell membrane.

Vitamins

Vitamins are a source of nutrients and have been included in most of the basal media prior to serum supplementation even though serum is a rich source of vitamins. Vitamins influence cell viability and proliferation and either essential for growth or added to stimulate growth. The B group vitamins are most commonly added for growth stimulation.

Trace Elements

Trace elements are often supplemented to serum-free media to replace those normally found in serum. These include such elements as zinc, copper, and selenium. Selenium is important due to its ability to remove harmful free radicals from the medium.

Serum

Serum is a very complex supplement containing mostly proteins but also growth factors, hormones, amino acids, sugars, trypsin inhibitors and lipids. The most common types of sera include fetal bovine (FBS), newborn calf (NCS), horse (Equine), pig (porcine), and human. FBS has been adopted as the standard supplement because of its rich content of growth factors and its low gamma globulin content

Buffering Systems

Maintaining the pH of the cell culture medium is critical to cell viability. Most cell lines grow well at 7.4 and are inhibited in growth at a pH of 6.8. Glucose is usually the sugar included in media and is metabolized by the cells very rapidly, at a rate faster than it is needed. Byproducts of this metabolism include pyruvic and lactic acids and CO2. One way to reduce the amount of lactic acid is to replace the glucose with other sugars such as galactose and fructose. These are utilized at a slower rate, but also result in a slower rate of cell growth. This replacement, however, can slow down the onset of a pH shift. The bicarbonate and carbon dioxide buffering system is most commonly used to maintain physiological pH (7.2-7.4) of a culture. Bicarbonate is a weak buffer with a pKa of 6.1 making a pH range of 7.2-7.6 more difficult to prevent rapid pH changes. Bicarbonate, however, is less expensive than other buffers, is non-toxic, and has nutritional value.

Organic systems

Organic buffers such as HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulphonicacid) do not require elevated levels of CO2 and provide an alternative to the use of bicarbonate for buffering.

Common Media Formulations

Alpha Modification of Eagle’s Medium (AMEM)

An enriched form of MEM, AMEM is a modification of Eagle’s Minimal Essential Medium that has been enriched with amino acids, vitamins, and nucleic acid precursors. Originally designed for hamster and mouse cell lines, AMEM with FBS is suitable for monolayer cultures.

Basal Media Eagle (BME)

BME is the simplest of the basal media and includes all the components essential for cell growth. Although not suitable for fastidious cell types, the medium may be used for immortal cell lines such as HeLa and primary mammalian fibroblasts.

Dulbecco’s Modification of Eagle’s Medium (DMEM)

DMEM is also an enriched form of BME as it contains four times the level of amino acids and vitamins. High and low glucose forms are available containing 4.5 g/L and 1.0 g/L of glucose, respectively. Low glucose DMEM is used for the maintenance of high-density cultures. DMEM was originally designed for mouse embryo cell lines.

DMEM/F-12

DMEM/F-12 is a 1:1 mixture of DMEM and Ham’s F-12 media. Originally formulated for rat neuroblastoma cells and MDCK cells, this medium is also suitable for some epithelial, endothelial, and granulose cell types.

Glasgow’s MEM

Glasgow’s MEM is a modification of MEM formulated to support the growth of BHK-21 cells. The medium was originally cased on BME with the addition of 10% tryptose phosphate broth and two times the concentration of amino acids and vitamins.

Ham’s F-10

Ham’s F-10 is optimized for clonal isolation and growth of cells. Originally formulated as a supplement containing albumin and fetuin, this medium supports the growth of human diploid cells, CHO cells, and human melanocytes.

Ham’s F-12

Ham’s F-12 is a modified form of Ham’s F-10 and contains an increased concentration of choline, inositol, putrescine, and several amino acids. Originally formulated as a serum free medium used in the cloning of CHO cells, Ham’s F-12 is also suitable for carcinoma cells, rat skeletal myoblasts, Chinese Hamster Liver cells, and rat, rabbit and chicken embryos

Iscove’s Modification of DMEM

Iscove’s is DMEM with additional amino acids and vitamins, also containing selenium and HEPES buffer. This is the first medium to contain HEPES for buffering capacity. Ferric nitrate normally included in DMEM is replaced with potassium nitrate in Iscove’s. Iscove’s is a highly enriched synthetic medium optimized for rapid proliferating, high cell density cultures. In addition of albumin and transferrin may be required, depending on the cell type.

Medium 199

Originally based on Earle’s balanced salt solution and formulated without protein, Medium 199 consists of nucleosides and nucleotides, as well as many amino acids, vitamins, growth factors, and lipids. Medium 199 is a complex early medium still widely used for the maintenance of non-transformed cells, vaccine, and virus production, and primary explants of epithelial cell. It was originally formulated to support the growth of chick embryo fibroblasts.

Rosewell Park Memorial Institute 1640

RPMI 1640 is a modification of McCoy’s 5A containing increased levels of inositol and is used primarily for various types of mammalian myeloma and hybridoma cells including mouse hybridomas, human leukocytes and B and T lymphocytes. The medium is also used for suspension cultures or monolayer human leukemic cells.MEDIA

Serum source of a Cell Culture

A sudden change in origin of serum can adversely affect the cell growth or can cause an undesirable change in the growth pattern of the cells. To avoid this we recommend the following protocol. *Note: If a cell line is fastidious, then it is recommended to follow proportion like 80:20, 60:40, 50:50, 40:60, 20:80 and 100% medium with new FBS.

Adaptation of Cell Cultures to a Serum-Free Medium (e.g. medium (SFM))

Sequential adaptation tends to be less harsh on cells than direct adaptation.

Adaptation with Conditioned Medium

An alternate method for adaptation to SFM involves the use of “conditioned medium.” This is medium the cells have been growing in for one full passage.

Facilitate adaptation as follows:

Passage 1

100% serum-supplemented medium

Passage 2

50% medium from passage 1: 50% SFM

Passage 3

50% medium from passage 2: 50% SFM

Passage 4

50% medium from passage 3: 50% SFM

Passage 5

100% SFM

Commonly Used Cell Lines in Cell Culture

CHO K1

VERO

BHK 21

MRC-5

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