Scientific Background and Utility of Serum-free Media in ELISPOT

Serum-free cell culture media allow users to standardize their cell culture conditions by avoiding the use of undefined and highly-variable serum products derived from humans or animals, e.g. human AB serum or fetal calf serum (FCS).

The high variability in the biological properties of different serum batches makes it necessary to pre-screen many batches in order to obtain a batch that is well-suited for a given application. Even a brief exposure of PBMC to a mitogenic serum batch, for example during washing or freezing of these cells, will result in a high background in cytokine assays. In addition, toxic or inhibitory serum batches will jeopardize the assay results. The unique performance of each serum batch necessitates the purchase of large lots to assure consistency of assay conditions as long as possible. This adds to the inconvenience and cost associated with the use of serum. Switching to or using a different batch of serum introduces a significant unpredictable variable to the test conditions each time, making test results difficult to compare. Furthermore, infectious risks associated with serum are leading to increasingly tighter restrictions for international shipments and exchange of any material that has been exposed to serum.

For all these reasons, there is considerable pressure from regulatory agencies and from the scientific community to avoid the use of serum and move to consistent, defined substitutes. However, because serum is naturally rich in a multitude of growth factors and other essentials for cell growth and functionality, it has been challenging to develop a serum substitute for primary cells in general, and for PBMC in particular. Most serum-free media contain enough additives to permit the growth of robust tumor cells, and hybridomas, but freshly-isolated human PBMC require more stringent conditions to maintain their viability and functionality in serum-free media.

CTL has been working for years with US governmental, industrial, and academic partners to standardize ELISPOT and other direct ex vivo cytokine assays performed on freshly-isolated or cryopreserved PBMC. These efforts have resulted in the CTL Serum-free Media Platform, which consists of three product lines that have been carefully designed to cover the four critical steps of working with human PBMC: washing, testing, freezing, and thawing. All four previously required the use of serum.

Low Background

Low background, or ideally no back- ground at all, is critical for PBMC-based T cell assays because rare antigen- specific T cells need to be detected amongst all other cells. The frequency of antigen-specific T cells seldom reaches one within a thousand PBMC, and often is as low as one within a hundred thousand, and less. Only in low-back- ground assays can such low-frequency T cells be reliably detected. Each batch of CTL Serum-free Media is quality controlled for low-background activity, typically providing zero to five non- specific IFN-γ, IL-2, IL-4, IL-5 and IL-17 ELISPOTs in PBMC of healthy donors. Serum, in contrast, frequently causes an elevated background (Figure 1A). Even brief exposure of PBMC to serum with mitogenic properties, e.g., during cryopreservation, or when washing the cells, can result in a high background. Entire T cell monitoring trials have been lost because PBMC were either frozen or tested with mitogenic serum!

High Signal

Serum always contains suppressive factors, such as TGFβ and IL-10. If present in increased concentrations, such molecules inhibit T cell activation, jeopardizing the detection of antigen- specific T cells in functional assays. For detecting antigen-specific CD8 cells, CTL Serum-free Medium matches or outperforms antigen-induced ELISPOT counts elicited with sera that have been qualified for T cell assays (Figure 1B). For CD4 cell detection, frequently remarkable increases in antigen- induced spot counts are seen with CTL Serum-free Media (Figure 2). This surprising finding is consistent with the notion that CD4 cells are more susceptible to suppressive serum cytokine effects than CD8 cells.

Standardization and Quality Control

Using different sera while working with PBMC is likely to produce different results. Each serum is a unique reagent containing unique concentrations of molecules that activate or inhibit T cells and APC. The need to work with serum has been a major obstacle for standardized and repro- ducible ex vivo experimentation. No longer! CTL Serum-free Media contain highly-defined and constant bioactive components, and each batch is quality controlled for consistant performance. The CTL Serum-free Media platform permits assay standardization within a laboratory, and even across laboratories (Figure 1).

Cost Effective

Consider the expense of working with serum-containing media, which far surpasses the cost of the serum itself. Add to that the work and materials involved in testing different batches to select the best performing one, then ordering in bulk quantities, taking up substantial freezer space to store long- term, and then preparation and sterile filterization of your self-made media and you have a substantial investment of time and money. Ready-to-use CTL Serum-free Media is a cost-effective, and hassle-free alternative.