The conversion of a particular cell or cell line from growth in serum-containing medium to serum-free medium is achieved through the weaning process. However, weaning is not required for all cell types. Rapid conversion of a cell population to serum-free conditions can be achieved by pelleting the cells and resuspending them in the serum-free medium. While this may be successful for some types of cells, a gradual conversion is more likely to yield the desired result.
Weaning is actually a process by which a subpopulation of cells that can proliferate in the absence of serum is selected. The degree of difficulty in selecting these cells is a function of the physical and nutritional requirements of the cells and the complexity of the serum-free formulation. Conversion of cells to growth in UltraCULTURETM Serum-free Medium can be relatively simple because it is a complex formula. Other formulations may contain reduced amounts of protein or be entirely devoid of proteins and peptides (i.e. UltraDOMA-PFTM Medium). In practice, these formulations require slightly more attention during the weaning process. However, the benefits of a low protein serum-free growth environment and subsequent reduction in downstream processing procedures more than offset the extra time spent in the weaning process.
Maintenance of cellular function is an aspect of the weaning process that must be monitored. One needs to ensure that the subpopulation selected exhibits the same characteristics with respect to cellular function as the population that was cultivated in the presence of serum. These functions are diverse and may include receptor expression, viral susceptibility, monoclonal antibody production, and recombinant gene expression. In many cases, an increase in product yield has been noted when cells are converted to a serum-free environment. However, each investigator should monitor the cellular function of interest to their application during the weaning process.
We recommend two protocols for the conversion of cell populations to a serum-free environment. These protocols may be used for mammalian and invertebrate cell types.
The first protocol may be used with attachment independent cells or cells that are loosely adherent and do not require trypsinization. It involves the gradual dilution of the serumcontaining medium with serum-free medium. The second protocol may be used with both attachment dependent and independent cell types and begins with the serum-free medium supplemented with serum. A gradual reduction in the serum concentration is performed at each subculture until serum-free growth is achieved. This latter protocol has the added advantage of establishing the limit of serum concentration for the cell type. Some cells (especially transfected lines) require small amounts of serum (i.e., 0.1–0.5% v/v). This method allows the investigator to titrate the serum to the lower limit.
The two weaning protocols are presented below. They represent our recommended procedures, however, each investigator may choose to make modifcations that better suit their particular application. In our experience, the minimum cell density maintained during the conversion process has a major effect on the outcome. We recommend that the cells be maintained above 3.0 × 105/mL for attachment independent and above 30% confluency for attachment dependent cells.