Until now, freezing and testing of PBMC has required the use of serum.

At isolation, PBMC need to make a critical transition from ideal in vivo conditions into the artificial tissue culture environment. Carefully selected, naturally rich serum has been essential for this transition.

Serum contains a plethora of bioactive molecules whose variable concentrations makes every serum batch unique. Careful testing of lots has been time-consuming yet necessary, and the limited size of each lot makes this testing a repeated inconvenience. In addition, working with serum is expensive. At the approximate cost of $1.000/L for human AB serum, complete RPMI medium made up in the lab is costly, and the need for sterile filtering each time adds to the expense. The cost is further increased by the need to purchase and store larger lots of a serum batch (Table 1).

From the good scientific/laboratory practice perspective, even an "ideal" serum batch is a unique reagent affecting T cell performance in vitro . The need to work with serum has been a major obstacle to reproducible and standardized ex vivo experimentation.

CTL has introduced media for serum-free work with PBMC

Unlike for tumor cell lines that have adapted to tissue culture conditions, ideally performing serum-free media for ex vivo human T cell work were not available. CTL's scientist have formulated a media portfolio that permits to perform serum free all steps of the ex vivo work with PBMC: the freezing, washing, and the testing of these cells. These media are:

• Convenient: avoiding the repeated effort for selecting "good" serum batches.
• Standardized: the same "serum substitute" can be used over and over, in the same and different laboratories.
• Cost effective: CTL's serum free media have been priced not to exceed the cost of traditional serum-containing media.
• Of ultimate importance: CTL's serum-free media outperforms even the "best" serum batches (Figure 1).

CTL's serum-free portfolio consists of a PBMC freezing medium, washing medium, and testing medium.

CTL freezing media: CTL-Cryo?/span>

When frozen according to our protocols, PBMC maintain their full functionality in ELISPOT assays (Table 2). The ability to freeze PBMC can be of major benefit, e.g. for clinical trials. First, instead of having to test the individual PBMC samples as they are obtained, they can be run economically in large batches. Second, samples from the same individuals obtained at different time points, and samples of individuals belonging to different groups can be run side-by-side, under identical conditions, avoiding inter-assay variability. Third, assay results can be reproduced, if necessary, on different aliquots from the same blood draw. Lastly, cryopreservation allows screening in a first assay for overall reactivity and then retesting of the sample to closer define the fine specificity, CD4/CD8 lineage, affinity, and cytokine profile of the specific T cells.

CTL washing media: CTL Wash?/span>

PBMC need to be washed after the Ficoll isolation before they are tested as fresh cells or are frozen. Washing is also required afterthawing the PBMC. Washing media typically contains serum because washing in saline or PBS alone reduces the PBMC's performance, and because the presence of proteins prevents cell clumping. However, even brief exposure of the PBMC to nonstandardized (e.g. mitogenic or toxic) serum will fundamentally affect the PBMC's performance. CTL's new serum-free washing media offers a standardized, convenient and cost-efficient alternative.

CTL testing media: CTL Test?/span>

When PBMC are used for immune monitoring, few antigen-specific T cells need to be detected amongst hundreds of thousands non relevant PBMC. Low background and strong signal resulting in maximal assay resolution are critical. Test media for such T cell assays have traditionally contained human AB serum that, frequently cause high background, or low signal. CTL's new serum-free testing media consistently provide lower noise and higher signal in such assays that even the "best" serum batches (Figure 1). They are standardized, convenient and cost-efficient.