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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