Top Down vs BottomUp Approaches to Serum Free Media Development

There are two basic approaches to developing serum-free media formulations. The first (top-down) involves taking an existing formulation for a similar cell line, supplemented with serum, and selecting constituents that are stimulatory for growth. This is followed by the slow reduction of serum in the medium. The second approach (bottom-up) involves the selection of a basal medium, analyzing individual components for their effects on growth, and combining them to yield a serum-free formulation.

Figure 1 Idealized growth response curve for a single nutrient.


Figure 1 Idealized growth response curve for a single nutrient.

The top-down approach is often easier to pursue since a working serum-free formulation can often be developed more quickly. Cell lines that belong to the same group, such as epithelial or transformed, often require the same growth factors for growth. Therefore, a formulation that works for one epithelial cell line may work for another with minimal modifications to certain growth factors or hormones (such as EGF, FGF, etc.). For this reason serum-free formulations can be designed faster by this approach.

However, the drawback to the top-down approach is that many components in the formulation may be unnecessary, and often inhibitory for growth. This can often result in the ''capping'' of the optimal performance of the medium (i.e., the maximum growth may not be achieved) as improvements are hindered by the presence of unwanted compounds.

The bottom-up approach, although more labor-intensive and time-consuming, can lead to higher-quality media. Only the components that are required for growth are included in the formulation, allowing for greater control in optimizing the medium. Thus, media developed in this way tend to have higher growth rates and are more easily improved since inhibitory compounds are less likely to be present.

Plackett-Burman Statistical Approach to Serum-Free Media Development

Another method of serum-free media development involves the use of a factorial experimental design, modeled after the Plackett-Burman statistical approach. This statistical method enables the systematic study of complex components and identifies nutritionally important factors for cell proliferation. In addition, the specific interactions between components (whether beneficial or detrimental) can be observed since the combination of nutrients is more important than the individual components by themselves. A large number of components can be studied at once to determine which factors, or combination of factors, are important for a serum-free formulation. This approach is not limited to cell growth studies and can also be valuable in determining which components in the medium are important for productivity, such as antibody production.

The steps involved in the approach are outlined as follows:

a. Select the basal medium and components that are to be analyzed for their ability to enhance growth or productivity.

b. Establish two concentrations for each component: a "high" concentration and a "low" concentration.

c. Grow the cells in the selected basal medium (e.g., DMEM) with serum (510%) in a well-plate culture. Allow the cells to initiate growth and division (-24 hr).

d. Remove the serum-medium and replace with the same basal medium plus combinations of media components (no serum), e.g., DMEM+insulin (high/low concentration)+transferrin (high/low concentration).

e. Include a positive control (serum-supplemented medium) and a negative control (medium with no components).

f. Count the viable cells (via MTT assay or trypan-blue exclusion method) in each well after -48 hr and/or assay for protein production (productivity).

g. Construct a matrix and determine which combinations of components, and at which concentration, are suitable for encouraging or sustaining cell growth (see Table 4).

h. Calculate the variances from all of the effects from the single factors and their interactions to determine which are significant. This will allow the determination of the best combination of components and thus the most suitable serum-free formulation.

i. Initiate an adaptation procedure for the cells to the new serum-free medium.

This stepwise approach has been utilized in many cases to design serum-free formulations. It has been attempted with the CHO cell line, where a serum-free

Table 4 Plackett-Burman Matrix


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