Cryopreservation of cells offers many advantages to the research community, such as banking of multiple aliquots of cells from multicenter studies of large cohorts of individuals. It allows precious samples to be available for future studies, often using newly developed techniques or assays. Additionally, samples of the same donor banked over time can be simultaneously processed, allowing greater inter- and intralaboratory control and reducing costs. But efficient cryopreservation of specimens is crucial for the success of these studies. It was previously shown that the viability of cryopreserved PBMCs has tremendous effects on the results of functional assays,13
so that at least 70% viability is necessary for conclusive responses to antigens and mitogens. PBMCs with viability of 70% can also be used for immunomagnetic cell separation, cytokine production studies, and flow cytometric analyses.33,34
Although cell recovery does not interfere with the results of immunologic assays, better recovery of only one proportion of cells in a heterogeneous population may preclude assay performance altogether. Based on these results, cryopreservation quality assurance programs are necessary for guaranteeing the quality of cryopreserved PBMCs and for increasing standardization between laboratories. For an efficient cryopreservation, FCS is used in most freezing media although each serum batch is unique in its ability to support optimal assay resolution and may potentially contain mitogenic or immune suppressive factors. It was already reported by The Cancer Vaccine Consortium of the Sabin Vaccine Institute that apparently the serum choice among their participants was responsible for suboptimal performance in one of their international ELISpot proficiency panels.35
However, to avoid import restrictions and negative impacts of viruses, growth factors and cytokines, human serum can be used for cryopreservation and immunologic assessment studies. But the cost of human serum is high and makes immunotherapy clinical trials, which are performed in large scale since the 1980s, unaffordable.
Because PBMC cryopreservation should be optimized and standardized to avoid affecting T-cell assays, we investigated the efficiency of novel serum-free and DMSO-reduced cryopreservation media by analyzing cell viability and recovery, as well as maintenance of T-cell functionality. Although some publications have recently been published describing the benefit of serum-free media in ELISpot assays with PBMCs cryopreserved in serum-containing media36,37
the culture medium for T-cells is in general still supplemented with serum.38–42
Additionally, we could detect significant differences in the outcome of ELISpot assays after PBMC cryopreservation in various FCS-based cryomedia, using pretested serum as medium supplement for ELISpot analysis (data not shown). As cryopreservation itself seems to influence the T-cell functionality considerably, independent of the medium used for PBMC cultivation and analysis, we focused our investigation on the replacement of FCS-containing cryomedia by serum-free compositions.
In this process, we could demonstrate that BSA fraction V is an appropriate candidate as a cryoprotectant to substitute the potentially immune-modulating FCS. Besides, with the addition of HES the reduction of the toxic DMSO concentration was possible, resulting only in a very slight decrease of the PBMC recovery, because the use of HES caused an increase of the glass transition temperature and the viscosity of the freezing medium.43
Extracellular cryoprotective agents, such as HES, usually withdraw the water and protect the cells from osmotically and electrically induced membrane damages from outside. In contrast, intracellular DMSO replaces the water shell in macromolecules, so that cells are able to survive intracellular ice crystallization.44,45
Moreover, adverse impacts on the function of antigen-specific lymphocytes at thawing were determined using IFN-γ ELISpot assay. At this, the focus was not on comparing the T-cell functionality between fresh isolated and cryopreserved PBMCs, but on demonstrating that T-cell response after cryopreservation in the new cryomedia was similar to FCS.
Our results were facilitated by other investigations that demonstrated that the function of cryopreserved PBMCs was associated with viability46
and that the type of protein additive in the cryopreservation media is critical for viability and antigen-specific function of T-cells.47
A multitude of studies already presented the disadvantageous effects of cryopreservation on the function of lymphocytes in antigen-specific T-cell assays. Cryopreservation of lymphocytes may have effects on cell surface molecules of T-cells such as CCR5 and CD45 RA/RO and may decrease responses to infectious diseases and recall antigens10
in both HIV-infected and noninfected volunteer donor blood. Further, cryopreservation can modify the ability of T-cells to secrete cytokines. Freezing and thawing cells hardly affected the cytokine secretion of cells from volunteer donors,15
and other investigators have demonstrated considerable differences after cryopreservation regarding the antigen-specific cytokine secretion of T-cells from cancer patients.18
The reliable evaluation of cryopreserved T-cell responses at time points distant from clinical trials is necessary for studies of immune-based therapies. The technology for sample analysis makes further progress, and adequate equipment and technical proficiency is not always available on site. Finally, an expanding number of patients across multiple sites will be necessary to conduct clinical trials, and consequently, analyzing T-cell responses on freshly isolated PBMCs will be impracticable.
On the basis of data described here, we developed 2 different serum-free cryopreservation media, one of them with a reduced amount of DMSO, produced under GMP conditions. Both cryomedia resulted in very good viability, recovery, and functionality of PBMCs after cryopreservation, compared to a commonly used FCS-containing cryomedium. The DMSO reduction by addition of HES revealed only marginal differences in the experimental outcome, indicating a promising approach for further improvements. Using these cryomedia, frozen samples can be transported without import restrictions, an important facilitation for clinical trials and in field studies. Besides, it is widely known that the use of serum in antigen-specific T-cell assays may lead to an increased unspecific activation of lymphocytes because of different cytokines and batch-dependent variability in results. Because of that, the serum-free and standardized cryomedia prevent time and cost intensive comparison of the efficiency of different FCS lots and increase reproducibility of results.
In future studies, the complete avoidance of animal proteins and products in combination with efficient cryopreservation SOPs should be the major aim. By minimizing the variety of components, reliability and standardization of results will be increased.