Skipping Serum in PBMC Workflows Holds Up in ELISpot Assays, Even After Two Years Frozen

For laboratories running enzyme-linked immunospot (ELISpot) assays in clinical trials, the media used at every step of handling blood cells have long been a quiet source of variability. Serum and serum-derived components such as fetal bovine serum (FBS) and bovine serum albumin (BSA) are conventionally added to wash buffers, freezing media, and culture medium, but each serum lot can behave differently, sometimes enhancing T-cell responses and sometimes suppressing them. Qualifying and bridging new lots consumes time and resources. A new study from researchers at Johnson & Johnson Innovative Medicine in Spring House, Pennsylvania, now maps out in detail what happens when serum is omitted at each stage of the workflow, from blood draw to the final spot count.

The research, published in AAPS Open, examined the impact of serum omission during peripheral blood mononuclear cell (PBMC) isolation, cryopreservation, and cell culture on T-cell viability and functionality in the context of clinical ELISpot assays. The team, led by Xinyuan Li with colleagues including Jaya Thomas, Tong-Yuan Yang, Michael Swanson, and Rashmi Jalah, deliberately built processing delays into their experiments to mimic conditions frequently encountered at clinical sites, where blood samples often travel long distances to laboratories equipped for cell isolation. Current guidance suggests blood should be processed into PBMCs and cryopreserved within eight hours of collection to accurately measure T-cell function, yet delays of one to two days are common in large multi-center studies.

The first set of experiments focused on the wash buffer used during PBMC isolation. Blood from four donors was processed with a next-day, 32-hour delay, using either phosphate-buffered saline (PBS) alone, PBS with 0.5 percent BSA, or PBS with 2 percent FBS. Adding BSA or FBS had no effect on pre-cryopreservation viability, but it changed yield dramatically: BSA increased PBMC yield by roughly 90 percent compared with PBS alone, while FBS increased it by about 40 percent. Despite this difference in recovery, ELISpot assay performance was indistinguishable among the three groups, whether cells were stimulated with a vehicle control or with the CEFX peptide pool, a positive control representing 176 peptide epitopes spanning a broad range of HLA subtypes and infectious agents.

The comparison extended to human serum albumin. When the researchers tested PBS alone against PBS with 0.5 percent BSA or PBS with 0.5 percent plasma-derived HSA, both albumin forms raised cell viability by about 15 percent and PBMC yield by roughly 100 percent. However, HSA strongly increased ELISpot assay background, indicating that components within the plasma-derived preparation interfered with downstream assay performance. The team then turned to a chemically defined recombinant HSA (rHSA), which performed cleanly: both BSA and rHSA consistently increased PBMC yield by about 50 percent without affecting assay performance. The authors conclude that BSA or rHSA can serve as key components of wash buffers to boost cell yield, whereas regular HSA should not be used during PBMC isolation for ELISpot workflows.

The second major question concerned long-term storage. Serum-free cryopreservation media such as CryoStor CS10 had previously been shown, in a large multi-center study, to match serum-containing freezing media when cells were cryopreserved within 24 hours and analyzed after relatively short storage. What remained unclear was whether serum-free cryomedium could preserve PBMC quality over the multi-year storage periods typical of clinical ELISpot studies. To address this, the researchers isolated PBMCs from five donors, cryopreserved them within eight hours in either serum-free CS10 or an in-house serum-containing medium called FBS10, consisting of 10 percent DMSO and 90 percent fetal bovine serum, and stored the vials in liquid nitrogen for two weeks, three months, six months, twelve months, and twenty-four months.

The results were striking. Sustained PBMC viability above 95 percent was observed after up to 24 months of liquid nitrogen storage with either cryomedium, and CS10 showed IFN-γ secretion in the ELISpot assay comparable to FBS10. The study therefore demonstrates that PBMC viability and functionality were maintained through at least two years of storage, and that omitting serum from the freezing medium had minimal impact on T-cell viability and functionality, even after two years in liquid nitrogen. This finding matters because cells in clinical trials are routinely banked for extended periods before analysis, and the earlier multi-center study had stored samples for an unspecified duration followed by two to twelve weeks at minus 80 degrees Celsius prior to analysis.

The third stage of the workflow, thawing and culture, revealed a more nuanced picture. Blood from four donors was processed with a BSA-containing wash buffer, cryopreserved in CS10 within four hours, and then thawed and cultured in either serum-containing RPMI plus FBS or serum-free AIM-V medium. Serum-free thawing medium did not significantly affect post-thaw viability, but post-thaw cell recovery dropped by about 20 percent with serum-free media, a statistically significant decrease. The researchers also observed lower ELISpot assay background when using serum-containing thawing and culture medium, even though responses to the CEFX positive control were similar between the two media. The authors note that this background effect appeared with a pre-screened FBS lot and caution that, because serum lots vary in composition and performance, the finding should not be generalized to all serum lots.

Processing delays compounded the culture-medium differences. Using donor-matched PBMCs isolated either within four hours or after a 52-hour delay, the team compared AIM-V, AIM-V supplemented with the BSA-based AlbuMAX supplement, and house-made RPMI plus FBS. For freshly processed samples, all three media performed equivalently. For delayed samples, however, ELISpot assay background rose by roughly tenfold in two of four donors when cells were cultured in AIM-V or AIM-V plus BSA, while serum-containing RPMI plus FBS showed no such heightened background. More importantly, the delayed samples lost antigen-specific functionality regardless of medium, and that loss could not be rescued by adding serum to the culture. The authors note that their earlier optimization work had already shown that granulocyte removal or deactivation, antioxidants, and cold temperature storage all failed to extend the processing window beyond 32 hours.

Taken together, the findings support a stage-specific strategy for clinical ELISpot studies. When samples are processed promptly, serum-free media perform comparably to serum-containing media across the workflow, offering advantages in standardization, reduced lot-to-lot variability, and regulatory compliance with chemically defined systems. When delays are anticipated, the authors recommend supplementing PBMC isolation wash buffers with BSA or rHSA to improve cell yield, using serum-free CS10 for cryopreservation given its demonstrated two-year stability, and using culture medium containing a pre-screened, qualified FBS lot to improve post-thaw recovery and reduce nonspecific background. These measures may mitigate selected effects of delayed processing but do not restore the antigen-specific response lost after a 52-hour delay, so processing windows, reagent lots, and stage-specific media should be predefined and qualified for the intended multi-site workflow.

The study involved healthy human donors who provided written informed consent, with the clinical protocol approved by an institutional review board. Samples were tested in duplicate or triplicate technical replicates, with reported results representing mean replicate values, and statistical comparisons used two-tailed paired t-tests or Mann-Whitney U-tests depending on data distribution. As with any donor-based study, the sample sizes of four to five donors per experiment and the observation that heightened background appeared in only two of four delayed donors limit generalization, and the authors note that further studies are needed to extend PBMC stability beyond 32 hours. Still, by systematically dissecting each stage of the workflow, the study offers clinical laboratories a practical, evidence-based map for choosing media that balance reproducibility, regulatory expectations, and the logistical realities of multi-center immune monitoring.

Subject of Research: Effects of omitting serum during PBMC processing, cryopreservation, and cell culture on clinical ELISpot assay performance

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

Article References: Li, X., Thomas, J., Yang, T.-Y., Swanson, M., & Jalah, R. (2026). Effects of serum omission during PBMC processing, cryopreservation, and cell culture on clinical ELISpot assay performance. AAPS Open, 12(1), Article 67. https://doi.org/10.1186/s41120-026-00207-w

Image Credits: AI Generated

DOI: 10.1186/s41120-026-00207-w

Keywords: ELISpot, PBMC, serum-free media, cryopreservation, fetal bovine serum, bovine serum albumin, IFN-gamma, T-cell viability, clinical immunology, sample processing delay, liquid nitrogen storage, AIM-V