Serum-Free Media

Although many cell lines are still propagated in medium supplemented with serum, in many instances cultures may now be propagated in serum-free media (Tables 10.1, 10.2). The need to (1) standardize media among laboratories, (2) provide specialized media for specific cell type, and (3) eliminate variable natural products, led to the development of more complex media, such as M199 of Morgan et al. [1950], CMRL 1066 of Parker et al. [1957], NCTC109 [Evans et al., 1956], Waymouth’s MB 572/1 [1959], NCTC135 [Evans & Bryant, 1965], and Birch and Pirt [1971] for L929 mouse fibroblast cells, and Ham’s F10 [1963] and F12 [1965] clonal growth media for Chinese hamster ovary (CHO) cells. Serum-free media were also developed for HeLa human cervical carcinoma cells [Blaker et al., 1971; Higuchi, 1977]. Although a degree of cell selectionmay have been involved in the adaptation of continuous cell lines to serum-free conditions, the MCDB series of media [Ham & McKeehan, 1978; see also Table 10.1], Sato’s DMEM/F12-based media [Barnes & Sato, 1980], and others based on RPMI 1640 [Carney et al., 1981; Brower et al., 1986; see also Table 10.2), demonstrated that serum could be reduced or omitted without apparent cell selection if appropriate nutritional and hormonal modifications were made to the media [Barnes et al., 1984a–d; Cartwright & Shah, 1994; Mather, 1998]. These also provided selective conditions for primary culture of particular cell types. Specific formulations (e.g., MCDB 110 [Bettger et al., 1981]) were derived to culture human fibroblasts [Ham, 1984], many normal and neoplastic murine and human cells [Barnes & Sato, 1980], lymphoblasts [Iscove & Melchers, 19 8], and several different primary cultures [Mather & Sato, 1979a,b; Sundqvist et al., 1991; Gupta et al., 1997; Keen & Rapson, 1995; Vonen et al., 1992] in the absence of serum, with, in several cases, some protein added [Tsao et al., 1982; Benders et al., 1991]. This list now covers a wide range of cell types, and many of the media are available commercially (see Appendix II). In addition, the need to remove animal proteins from the in vitro production of biopharmaceuticals has generated a number of formulations for continuous cell lines such as CHO and hybridomas [Froud, 1999; Ikonomou et al., 2003; Shah, 1999], not least for some of the safety issues involved [Merten, 1999].

10.1 DISADVANTAGES OF SERUMUsing serum in a medium has a number of disadvantages:(1) Physiological Variability. The major constituents of serum, such as albumin and transferrin, are known, but serum also contains a wide range of minor components that may have a considerable effect on cell growth (see Table 9.5). These components include nutrients (amino acids, nucleosides, sugars, etc.), peptide growth factors, hormones, minerals, and lipids, the concentrations and actions of which have not been fully determined.(2) Shelf Life and Consistency. Serum varies from batch to batch, and at best a batch will last one year, perhaps deteriorating during that time. It must then be replaced with another batch that may be selected as similar, but will never be identical, to the first batch.(3) Quality Control. Changing serum batches requires extensive testing to ensure that the replacement is as close as possible to the previous batch. This can involve several tests (for growth, plating efficiency, and special functions; see Section 9.7.2) and a number of different cell lines.

(4) Specificity. If more than one cell type is used, each type may require a different batch of serum, so that several batches must be held on reserve simultaneously. Coculturing different cell types will present an even greater problem.(5) Availability. Periodically, the supply of serum is restricted because of drought in the cattle-rearing areas, the spread of disease among the cattle, or economic or political reasons. This can create problems at any time, restricting the amount of serum available and the number of batches to choose from, but can be particularly acute at times of high demand. Today, demand is increasing, and it will probably exceed supply unless the majority of commercial users are able to adopt serum-free media. Although an average research laboratory may reserve 100–200 L of serum per year, a commercial biotechnology laboratory can use that amount or more in a week.(6) Downstream Processing. The presence of serum creates a major obstacle to product purification and may even limit the pharmaceutical acceptance of the product.(7) Contamination. Serum is frequently contaminated with viruses, many of which may be harmless to cell culture but represent an additional unknown factor outside the operator’s control [Merten, 1999]. Fortunately, improvements in serum sterilization techniques have virtually eliminated the risk of mycoplasma infection from sera from most reputable suppliers, but viral infection remains a problem, despite claims that some filters may remove viruses (Pall Gelman). Because of the risk of spreading bovine spongiform encephalitis among cattle, cell cultures and serum shipped to the United States orAustralia require information on the country of origin and the batch number of the serum. Serum derived from cattle in New Zealand probably has the lowest endogenous viral contamination, as many of the viruses found in European and North American cattle are not found in New Zealand.(8) Cost. Cost is often cited as a disadvantage of serum supplementation. Certainly, serum constitutes the major part of the cost of a bottle of medium (more than 10 times the cost of the chemical constituents), but if it is replaced by defined constituents, the cost of these may be as high as that of the serum. However, as the demand for such items as transferrin, selenium, insulin, etc., rises, the cost is likely to come down with increasing market size, and serum-free media will become relatively cheaper. The availability of recombinant growth factors, coupled with market demand, may help to reduce their intrinsic cost.(9) Growth Inhibitors. As well as its growth-promoting activity, serum contains growth-inhibiting activity, and although stimulation usually predominates, the net effect of the serum is an unpredictable combination of both inhibition and stimulation of growth. Although substances such as PDGF may be mitogenic to fibroblasts, other constituents of serum can be cytostatic. Hydrocortisone, present at around 1 × 10−8 M in fetal serum, is cytostatic to many cell types, such as glia [Guner et al., 1977] and lung epithelium [McLean et al., 1986], at high cell densities (although it may be mitogenic at low cell densities), and TGF-β, released from platelets, is cytostatic to many epithelial cells.(10) Standardization. Standardization of experimental and production protocols is difficult, both at different times and among different laboratories, because of batch-tobatch variations in serum.

10.2 ADVANTAGES OF SERUM-FREE MEDIA

All of the above problems can be eliminated by removal of serum and other animal products. Serum-free media have, in addition, two major positive benefits.

10.2.1 Selective MediaOne of the major advantages of the control over growth promoting activity afforded by serum-free media is the ability to make a medium selective for a particular cell type (see Tables 10.1 and 10.2). Fibroblastic overgrowth can be inhibited in breast and skin cultures by using MCDB 170 [Hammond et al., 1984] and 153 [Peehl and Ham, 1980], melanocytes can be cultivated in the absence of fibroblasts and keratinocytes [Naeyaert et al., 1991], and separate lineages and even stages of development may be selected in hematopoietic cells by choosing the correct growth factor or group of growth factors (see Section 23.4). Many of these selective media are now available commercially along with cultures of selected cell types (see Table 10.4).

10.2.2 Regulation of Proliferation and DifferentiationAdd to the ability to select for a specific cell type the possibility of switching from a growth-enhancing medium for propagation to a differentiation-inducing medium by altering the concentration and types of growth factors and other inducers.

10.3 DISADVANTAGES OF SERUM-FREE MEDIASerum-free media are not without disadvantages:(1) Multiplicity of Media.

Each cell type appears to require a different recipe, and cultures from malignant tumors may vary in requirements from tumor to tumor, even within one class of tumors. Although this degree of specificity may be an advantage to those isolating specific cell types, it presents a problem for laboratories maintaining cell lines of several different origins.

(2) Selectivity.Unfortunately, the transition to serum-free conditions, however desirable, is not as straightforward as it seems. Some media may select a sublineage that is not typical of the whole population, and even in continuous cell lines, some degree of selection may still be required. Cells at different stages of development (e.g., stem cells vs. committed precursor cells) may require different formulations, particularly in the growth factor and cytokine components.

(3) Reagent Purity. The removal of serum also requires that the degree of purity of reagents and water and the degree of cleanliness of all apparatus be extremely high, as the removal of serum also removes the protective, detoxifying action that some serum proteins may have. Although removing this action is no doubt desirable, it may not always be achievable, depending on resources.

(4) Cell Proliferation. Growth is often slower in serumfree media, and fewer generations are achieved with finite cell lines.

(5) Availability. Although improving steadily, the availability of properly quality-controlled serum-free media is quite limited, and the products are often more expensive than conventional media.