The Case for Serum-Free Media Technical Brief
Sheldon E. Broedel, Jr., Ph. D.
Athena Environmental Sciences, Inc., Baltimore , MD
Why Use Serum-Free Media?
The use of serum-free medium and its related genera of animal-free
and protein-free media has grown significantly in the last 15 years. This is
particularly true in industrial applications where the use of serum presents
a safety hazard as well as a source of unwanted contamination for the production
of biopharmaceuticals. Serum-free medium is prepared without the use of animal
serum, but may contain serum constituents or substitutes thereof. Animal-free
medium is similar to serum-free except that the components are derived from
non-animal sources. Recombinant proteins replace native proteins and the
nutrients are obtained from synthetic, plant or microbial sources. In contrast,
protein-free medium is defined as devoid of protein, although few formulations
of these media can be 100% protein-free without loss of function. A better
definition would be low protein where minimal quantities of small mass proteins
are employed.
The three types of media can be thought of as gradations of complexity.
Serum-free media is the more complex composition designed for universal use in
culturing mammalian cell lines. Animal-free and protein-free media formulations
are less complex and more defined, but are limited to the cultivation of specific
cell types. Thus, the selection of one medium over another will depend on the
intended use. For most research applications, serum-free medium is the best
choice as it provides broad-spectrum utility while giving good control of the
composition. In contrast, in the manufacture of protein-based drugs, potential
contaminants and production costs play a more significant role in the selection
of a more defined medium composition.
Serum-free medium has four basic advantages over serum containing
medium. These include: 1] a simplified and better defined composition, 2] a
reduced degree of contaminants, 3] elimination of a potential source of
infectious agents, and 4] lower cost.
Simplified and Defined Composition
Serum is an ill defined component of medium. Typically used at 5% to
10% v/v, serum provides a range of factors which have proved necessary for the
cultivation of mammalian and insect cells. Serum however can be derived from
different sources and the composition of the serum can vary greatly. Advances
in the manufacture and processing of serum has reduced this variability, but
many researchers continue to find it necessary to qualify new lots of serum by
comparing the growth of their cell lines in the new serum relative to a current
lot. With a typical acceptance criteria of ?20%, the variance of whatever
physiological function is being measured can be quite high. This leads to poor
reproducibility of results between and within laboratories and stories abound
of irreproducible results which can be directly attributed to the use of serum
as a media component. A quantitative understanding of cellular physiology cannot
be obtained with such ill-defined and variable conditions. Serum-free medium on
the other hand is a more defined medium. While composed of many constituents,
the composition is known and the level of each component precisely defined.
Therefore, the variance seen with serum containing medium is eliminated giving
a more controlled environment in which cells can be grown.
Reduced Range and Level of Contaminants
Medium prepared with serum at 10% has a protein concentration of 6,200
to 10,000 mg/L. A typical recombinant protein produced in mammalian cells is
anywhere from a few mg/L to 1,000 mg/L. For a native protein, the level of
accumulation can be even lower. The serum proteins thus become a major contaminant
of any crude supernatant in which the target protein accumulates. Further, if the
target protein is functionally, biochemically or physically related to a serum
protein, then it may prove difficult to separate the target protein from the serum
protein. This could be disastrous for the production of a protein-based drug, but
equally troubling to the researcher trying to study the functional and physical
properties of the target protein. The protein concentration of serum-free media
on the other hand is between 50 mg/L and 1,000 mg/L. Unlike serum, the composition
is known and typically three proteins, albumin, transferin and insulin, comprise
80% to 90% of the proteins present. Consequently, the relative level of the target
protein is significantly higher and with fewer contaminants. Thus, the target
protein is easier to purify in fewer steps which gives higher recovery values.
Drug discovery, physiological, or gene expression studies are simpler
to perform with serum-free medium. Serum components are known to bind, degrade
or otherwise interact with chemicals added to the medium. Complex associations
are possible between the serum, the added effector and the cells. Thus, the
effect elicited by the added chemical can be altered or eliminated by the
serum factors. Serum-free medium by contrast has fewer possible interferents
and where an interaction is observed, more readily controlled.
Elimination of Potential Source of Infectious Agents
Viral, bacterial and fungal contamination of serum has for some years
been a concern by manufacturers of biopharmaceuticals. This has been a driving
force behind the adoption of serum-free, animal-free and protein-free media
formulations in the manufacturing process. More recently, transmissible spongiform
encephalophathies described in animal-derived materials has lead to the elimination
of any animal product for the manufacture of a pharmaceutical product. In the
research setting, where more fastidious cell lines are employed, the complete
elimination of animal-derived components is both impractical and unusable.
Recombinant forms of some key components remain expensive and in some cases not
available. Many of the commercially available animal-free and protein-free media
were designed for specific cell types and will not support the growth of a wide
range of cell types. Because the materials used for the production of serum-free
media are for the most part highly purified, the risk of contaminating a culture
with adventitious agents is greatly reduced or eliminated altogether. For
non-pharmaceutical applications, serum-free medium remains a good balance
between safety and efficacy.
Lower Cost and Availability
Serum can cost between $7 and $50 per liter of medium depending on the
type and percentage of serum used. Fetal calf serum, the most commonly used source
of serum, is also the most costly, with current prices (August 2002) averaging $456/L
of serum. Therefore, serum can contribute significantly to the cost of the medium.
Serum-free medium by contrast averages about $120/L with a range from $26/L for a
DMEM derivative to $692/L for highly specialized bone marrow medium. Most of the
serum-free media are supplied ready-to-use, so they are available without the need of
preparing a multi-component reagent.
Disadvantages
The use of serum-free medium is routine for many cell types and many
formulations are available in the literature or through a number of vendors.
Nevertheless, there are a few drawbacks that should be considered when using
serum-free medium. First, an investment in time is required to adopt a particular
cell line to serum-free medium. The cells will have to be weaned from serum
slowly. Moreover, some cell lines may require the addition of growth factors
specific to that cell type to overcome a deficiency in the particular medium
employed. It is advisable to begin with a serum-free medium which has a source
of growth factors, such as pituitary extract, which can be incrementally removed
if necessary.
The second limitation is that the low protein concentration of serum-free
medium while an advantage for reducing potential sources of contamination removes
proteins which play a role in shear protection and attachment to growth substratum.
The BSA present in serum protects cells grown in suspension from shear damage. The
addition of Pluronic F68 or polyethylene glycol may be needed in place of BSA. BSA
also provides other transport functions and the replacement of native sources of
albumin with recombinant sources is not straightforward. Alternative supplements
may be needed. Many of these issues are resolved by the commercially available
formulation and only become a concern when developing a custom medium.
Attachment-dependent cell lines require an extracellular matrix on the
growth substratum. Serum provides some the components for this matrix. Therefore,
when using serum-free medium the substratum (plastic dishes) should be pre-coated
with a fibronectin, laminin or another suitable alternative such as FNC Coating Mix®
(a fibronectin/collagen mixture manufactured by AthenaES, Baltimore, MD), Pronectin®
(a synthetic fibronectin polymer manufactured by Sanyo Chemical Industries, Kyoto,
Japan) or Matrigel.
Summary
Serum-free medium is an excellent alternative to standard serum-containing
media for the cultivation of cells. It has several marked advantages which include
better definition of the composition, reduced contamination by adventitious and
infectious agents, and lower cost. Further, the commercial availability of many
variations of serum-free media has made it easy to obtain and employ. Consequently,
the use of serum-free medium has now become a routinely used reagent in many
laboratories for the culture of a wide variety of cell types.
About the Author
Sheldon E. Broedel, Jr., Ph.D. serves as the Chief Science Officer at
AthenaES? . He has over 23 years of research experience, 15 of which were spent
in the biotechnology industry. Dr. Broedel has broad expertise in a range of
biology disciplines with over 20 products and patents to his credit. Currently,
his research team is developing reagents designed to increase the production of
recombinant proteins.
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