Dr. Tarek Elbashiti Assoc. Prof. of Biotechnology Serum-Free Media Dr. Tarek Elbashiti Assoc. Prof. of Biotechnology

Although many cell lines are still propagated in medium supplemented with serum, in many instances cultures may now be propagated in serum-free media. 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.

Although a degree of cell selection may have been involved in the adaptation of continuous cell lines to serum-free conditions, the MCDB series of media, Sato’s DMEM/F12-based media, and others based on RPMI 1640, demonstrated that serum could be reduced or omitted without apparent cell selection if appropriate nutritional and hormonal modifications were made to the media.

DISADVANTAGES OF SERUM 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. (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) 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. 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. 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.

(8) Cost: 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. 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. Hydrocortisone, present at around 1×10−8 M in fetal serum, is cytostatic (Inhibiting cell growth and division) to many cell types, such as glia and lung epithelium, 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-to batch variations in serum.

ADVANTAGES OF SERUM-FREE MEDIA 1. Selective Media One 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. Fibroblastic overgrowth can be inhibited in breast and skin cultures by using MCDB 170 and 153. Many of these selective media are now available commercially along with cultures of selected cell types.

2. Regulation of Proliferation and Differentiation Add 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.

DISADVANTAGES OF SERUM-FREE MEDIA 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: Some media may select a sub-lineage 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.

(4) Cell Proliferation: Growth is often slower in serum-free 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.

REPLACEMENT OF SERUM The essential factors in serum have been described and include: adhesion factors such as fibronectin; (2) peptides, such as insulin, PDGF, and TGF-β, that regulate growth and differentiation; (3) Essential nutrients, such as minerals, vitamins, fatty acids, and intermediary metabolites; and (4) hormones, such as insulin, hydrocortisone, estrogen, and triiodothyronine, that regulate membrane transport, phenotypic status, and the constitution of the cell surface.

SELECTION OF SERUM-FREE MEDIUM If the reason for using a serum-free medium is to promote the selective growth of a particular type of cell, then that reason will determine the choice of medium (e.g., MCDB 153 for epidermal keratinocytes, LHC-9 for bronchial epithelium, HITES for small-cell lung cancer, MCDB 130 for endothelium, etc.; see Tables 10.1, 10.2 and 10.4).

Commercially Available Serum-Free Media Several suppliers (see Table 10.4 and Appendix II) now make serum-free media. Some are defined formulations, such as MCDB 131 (Sigma) for endothelial cells and LHC-9 (Biosource International) for bronchial epithelium, whereas others are proprietary formulations, such as CHO-S-SFM for CHO-K1 cells and Opti-MEM for hematopoietic cells (Invitrogen). Many are designed primarily for culture of hybridomas, when the formation of a product that is free of serum proteins is clearly important, but others are applicable to other cell types.

Adaptation to Serum-Free Media Adaptation to serum-free medium is usually carried out over several serial subcultures, with the serum concentration being reduced gradually. Once stable cell proliferation is established at one concentration, subculture cells into a lower concentration until stable growth is reestablished and then dilute the serum again. In suspension cultures, this is done by monitoring the viable cell count and diluting the cell suspension accordingly, perhaps keeping the minimal cell concentration higher than for normal subculture.

For monolayer cultures, reduce the serum concentration a few days before subculture, and then subculture into the new low serum concentration. During the adaptation process it may be necessary to supplement the medium with factors known to replace serum.