Effects of serum omission during PBMC processing, cryopreservation, and cell culture on clinical ELISpot assay performance

Serum is often used as an additive for cryopreservation media, wash buffers, and cell culture medium. As serum lots can have variable performance, serum-free variants are desirable alternatives. This research paper investigates the impact of omitting serum from wash buffer, cryomedium, or cell culture medium on T-cell viability and functionality in the context of conducting clinical ELISpot assays. Although serum-free media show comparable assay performance as serum-containing media in ELISpot assays, the impact of serum omission on PBMC functionalities from delayed sample processing is unclear. In addition, although serum-free freezing media demonstrate comparable viability and IFN-γ secretion measured in ELISpot assay from PBMCs as serum-containing cryopreservation media, the effects of serum omission on PBMC functionality from prolonged storage in liquid nitrogen remain unclear. The study finds that the inclusion of bovine serum albumin in wash buffer during PBMC isolation increases yield of overall PBMC cells. In addition, fetal bovine serum-containing cell culture medium reduces assay background, especially when there is a delay of PBMC isolation. Moreover, we found minimal impact on T-cell viability and T-cell functionality when omitting serum in cryopreservation of PBMCs, even after 2-year storage in liquid nitrogen. Collectively, these findings provide strategies into selecting appropriate media for clinical ELISpot studies, optimizing and improving clinical operation processes and compliance regarding sample processing and shipment.