Selecting the correct medium and serum in Animal Biotechnology

Selecting the medium and serum in animal Biotechnology

Most of the media described in our previous post- Serum – as a growth medium in animal tissue culture were developed to support particular cell lines or conditions. Many were developed with L929 mouse fibroblasts or HeLa cervical carcinoma cells, and Ham’s F12 was designed for Chinese hamster ovary (CHO) cells; all now have more general applications and have become classic formulations. Among them, data from suppliers would indicate that RPMI 1640, DMEM, and MEM are the most popular, making up about 75% of sales. Other formulations seldom account for more than 5% of the total; most constitute 2–3%, although blended DMEM/F12 comes closer, with over 4%. Eagle’s Minimal Essential Medium (MEM) was developed from Eagle’s Basal Medium (BME) by increasing the range and concentration of the constituents. For many years, Eagle’s MEM had the most general use of all media. Dulbecco’s modification of BME (DMEM) was developed for mouse fibroblasts for transformation and virus propagation studies. It has twice the amino acid concentrations of MEM, has four times the vitamin concentrations, and uses twice the HCO₃ −and CO₂ concentrations to achieve better buffering. α-MEM has additional amino acids and vitamins, as well as nucleosides and lipoic acid; it has been used for a wide range of cell types, including hematopoietic cells. Ham’s F12 was developed to clone CHO cells in low-serum medium; it is also used widely, particularly for clonogenic assays and primary culture. CMRL 1066, M199, and Waymouth’s media were all developed to grow L929 cells serum-free but have been used alone or in combination with other media, such as DMEM or F12, for a variety of more demanding conditions. RPMI1640 and Fischer’s media were developed for lymphoid cells—Fischer’s specifically for L5178Y lymphoma, which has a high folate requirement. RPMI 1640 in particular has quite widespread use, often for attached cells, despite being designed for suspension culture and lacking calcium. L15 medium was developed specifically to provide buffering in the absence of HCO₃ − and CO₂. It is often used as a transport and primary culture medium for this reason, but its value was diminished by the introduction of HEPES and the demonstration that HCO₃ − and CO₂ are often essential for optimal cell growth, regardless of the requirement for buffering.

Information regarding the selection of the appropriate medium for a given type of cell is usually available in the literature in articles on the origin of the cell line or the culture of similar cells. Information may also be obtained from the source of the cells. Cell banks, such as ATCC and ECACC, provide information on media used for currently available cell lines, and data sheets can be accessed from their websites. Failing this, the choice is made either empirically or by comparative testing of several media, as for selection of serum.

Many continuous cell lines (e.g., HeLa, L929, BHK21), primary cultures of human, rodent, and avian fibroblasts, and cell lines derived from them can be maintained on a relatively simple medium such as Eagle’s MEM, supplemented with calf serum. More complex media may be required when a specialized function is being expressed or when cells are sub-cultured at low seeding density (<1×10³ /mL), as in cloning. Frequently, the more demanding culture conditions that require complex media also require foetal bovine serum rather than calf or horse serum, unless the formulation specifically allows for the omission of serum.

If information is not available, a simple cell growth experiment with commercially available media and multiwell plates can be carried out in about two weeks. Assaying for clonal growth and measuring the expression of specialized functions may narrow the choice further [you may soon find the protocol for this cell growth experiment in our upcoming posts, or just mail us at BiotechExplorer@gmail.com].

Autoclavable media are available from commercial suppliers (you may search it on web). They are simple to prepare from powder and are suitable for many continuous cell strains. They may need to be supplemented with glutamine for most cells and usually require serum.

Note a simple trick: The cost of serum should be calculated on the basis of the volume of the medium when cell yield is not important, but if the objective is to produce large quantities of cells, one should calculate serum costs on a per-cell basis. Thus, if a culture grows to 1×10⁶ /mL in serum A and 2×10⁶ mL in serum B, serum B becomes the less expensive by a factor of two, given that product formation or some other specialized function is the same.

If foetal bovine serum seems essential, try mixing it with calf serum. This may allow you to reduce the concentration of the more expensive foetal serum. If you can, leave out serum altogether, or reduce the concentration, and use a serum-free formulation.

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Did you read these few relative topics ?

Development of media for animal tissue culture

Serum – as a growth medium in animal tissue culture

Complete Media - Animal tissue culture

Use of Antibiotics in Animal Tissue Culture for maintaining sterility

Balanced Salt Solution in Animal Tissue Culture

Animal Biotechnology - Tissue Culture and Cell Culture 

Maintenance of Sterility in Animal Tissue Culture Labs

Serum – as a growth medium in animal tissue culture

Serum – as a growth medium in animal tissue culture

Serum contains growth factors, which promote cell proliferation, and adhesion factors and antitrypsin activity, which promote cell attachment. Serum is also a source of minerals, lipids, and hormones, many of which may be bound to protein. The sera (plural form of 'serum') used most in tissue culture are bovine calf, foetal bovine, adult horse, and human serum. Calf serum (CS) and foetal bovine (FBS) serum are the most widely used, the latter particularly for more demanding cell lines and for cloning. Human serum is sometimes used in conjunction with some human cell lines, but it needs to be screened for viruses, such as HIV and hepatitis B. Horse serum is preferred to calf serum by some workers, as it can be obtained from a closed donor herd and is often more consistent from batch to batch. Horse serum may also be less likely to metabolize polyamines, due to lower levels of polyamine oxidase; polyamines are mitogenic for some cells.

Proteins

Although proteins are a major component of serum, the functions of many proteins in vitro remain obscure; it may be that relatively few proteins are required other than as carriers for minerals, fatty acids, and hormones. Those proteins for which requirements have been found are albumin, which may be important as a carrier of lipids, minerals, and globulins; fibronectin (cold-insoluble globulin), which promotes cell attachment, although probably not as effectively as cell-derived fibronectin; and α 2-macroglobulin, which inhibits trypsin. Fetuin in foetal serum enhances cell attachment and transferrin binds iron, making it less toxic and bioavailable. Other proteins, as yet uncharacterized, may be essential for cell attachment and growth.

Protein also increases the viscosity of the medium, reducing shear stress during pipetting and stirring, and may add to the medium’s buffering capacity.

Growth Factors

Natural clot serum stimulates cell proliferation more than serum from which the cells have been removed physically (e.g., by centrifugation). This increased stimulation appears to be due to the release of platelet-derived growth factor (PDGF) from the platelets during clotting. PDGF is one of a family of polypeptides with mitogenic activity and is probably the major growth factor in serum. PDGF stimulates growth in fibroblasts and glia, but other platelet-derived factors, such as TGF-β, may inhibit growth or promote differentiation in epithelial cells. Other growth factors, such as fibroblast growth factors (FGFs), epidermal growth factor (EGF), endothelial cell growth factors such as vascular endothelial growth factor (VEGF) and angiogenin, and insulin-like growth factors IGF-I and IGF-II, which have been isolated from whole tissue or released into the medium by cells in culture, have varying degrees of specificity and are probably present in serum in small amounts. Many of these growth factors are available commercially as recombinant proteins, some of which also are available in long-form analogues (Sigma) with increased mitogenic activity and stability.

Hormones

Insulin promotes the uptake of glucose and amino acids and may owe its mitogenic effect to this property or to activity via the IGF-I receptor. IGF-I and IGF-II bind to the insulin receptor, but also have their own specific receptors, to which insulin may bind with lower affinity. IGF-II also stimulates glucose uptake. Growth hormone may be present in serum—particularly foetal serum—and, in conjunction with the somatomedins (IGFs), may have a mitogenic effect.

Hydrocortisone is also present in serum—particularly foetal bovine serum—in varying amounts and it can promote cell attachment and cell proliferation, but under certain conditions (e.g., at high cell density) may be cytostatic and can induce cell differentiation.

Nutrients and Metabolites

Serum may also contain amino acids, glucose, Oxo (keto) acids, nucleosides, and a number of other nutrients and intermediary metabolites. These may be important in simple media but less so in complex media, particularly those with higher amino acid concentrations and other defined supplements.

Lipids

Linoleic acid, oleic acid, ethanolamine, and phospho-ethanol amine are present in serum in small amounts, usually bound to proteins such as albumin.

Minerals

Serum replacement experiments have also suggested that trace elements and iron, copper, and zinc may be bound to serum protein. McKeehan demonstrated a requirement for selenium, which probably helps to detoxify free radicals as a cofactor for GSH synthetase.

Inhibitors

Serum may contain substances that inhibit cell proliferation. Some of these may be artefacts of preparation (e.g., bacterial toxins from contamination before filtration, or antibodies, contained in the γ-globulin fraction, that cross-react with surface epitopes on the cultured cells), but others may be physiological negative growth regulators, such as TGF-β. Heat inactivation removes complement from the serum and reduces the cytotoxic action of immunoglobulin without damaging polypeptide growth factors, but it may also remove some more labile constituents and is not always as satisfactory as untreated serum.

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Did you read these few relative topics ?

Development of media for animal tissue culture

Complete Media - Animal tissue culture

Use of Antibiotics in Animal Tissue Culture for maintaining sterility

Balanced Salt Solution in Animal Tissue Culture

Animal Biotechnology - Tissue Culture and Cell Culture 

Maintenance of Sterility in Animal Tissue Culture Labs

Complete Media - Animal tissue culture

The term complete medium implies a medium that has had all its constituents and supplements added and is sufficient for the use specified. It is usually made up of a defined medium component, some of the constituents, such as glutamine, may be added just before use, and various supplements, such as serum, growth factors, or hormones.

Defined media range in complexity from the relatively simple Eagle’s MEM, which contains essential amino acids, vitamins, and salts, to complex media such as medium 199 (M199), CMRL 1066, MB 752/1, RPMI 1640, and F12 and a wide range of serum-free formulations. The complex media contain a larger number of different amino acids, including nonessential amino acids and additional vitamins, and are often supplemented with extra metabolites (e.g., nucleosides, tri carboxylic acid cycle intermediates, and lipids) and minerals. Nutrient concentrations are, on the whole, low in F12 (which was optimized by cloning) and high in Dulbecco’s modification of Eagle’s MEM (DMEM), optimized at higher cell densities for viral propagation. Barnes and Sato [in 1980] used a 1:1 mixture of DMEM and F12 as the basis for their serum-free formulations to combine the richness of F12 and the higher nutrient concentration of DMEM. Although not always entirely rational, this combination has provided an empirical formula that is suitable as a basic medium for supplementation with special additives for many different cell types.

 

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Did you read these ?

Development of media for animal tissue culture

Use of Antibiotics in Animal Tissue Culture for maintaining sterility

Balanced Salt Solution in Animal Tissue Culture

Animal Biotechnology - Tissue Culture and Cell Culture 

Maintenance of Sterility in Animal Tissue Culture Labs