Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols

Abstract

Robust and sensitive ELISPOT protocols are commonly applied concomitant with the development of new immunotherapeutics. Despite the knowledge that individual serum batches differ in their composition and may change properties over time, serum is still commonly used in immunologic assays. Commercially available serum batches are expensive, limited in quantity and need to be pretested for suitability in immunologic assays, which is a laborious process. The aim of this study was to test whether serum-free freezing media can lead to high cell viability and favorable performance across multiple ELISPOT assay protocols. Thirty-one laboratories from ten countries participated in a proficiency panel organized by the Cancer Immunotherapy Immunoguiding Program to test the influence of different freezing media on cell quality and immunologic function. Each center received peripheral blood mononuclear cells which were frozen in three different media. The participants were asked to quantify antigen-specific CD8+ T-cell responses against model antigens using their locally established IFN-gamma ELISPOT protocols. Self-made and commercially available serum-free freezing media led to higher cell viability and similar cell recovery after thawing and resting compared to freezing media supplemented with human serum. Furthermore, the test performance as determined by (1) background spot production, (2) replicate variation, (3) frequency of detected antigen-specific spots and (4) response detection rate was similar for serum and serum-free conditions. We conclude that defined and accessible serum-free freezing media should be recommended for freezing cells stored for subsequent ELISPOT analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-012-1359-5) contains supplementary material, which is available to authorized users.

The importance of correctly timing cancer immunotherapy

“…Therefore, an important aspect is to ensure that the measurement and reporting of the results obtained are comparable between laboratories so that it is easier to interpret and value the data generated. In the last decade a huge effort, undertaken by the Association of Cancer ImmunoTherapy's (CIMT's) immunoguiding program [125] and Cancer Immunotherapy Consortium (CIC) [126], has focused on this in the realm of human studies, and this has successfully led to: (i) a strong awareness of the need to harmonize or standardize measurements, (ii) to technical improvements and harmonization of the assays used, and (iii) to more transparent reporting [127][128][129]. It can be envisaged that similar efforts by scientists performing studies in mouse tumor models will lead to a quicker design of immunotherapeutic strategies, which expedite the translation to the treatment of patients.…”

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8+ T cells with detection by ELISPOT and HLA-multimer staining

“…ELISPOT assay and multimer staining, have been the subject of a number of harmonisation studies, in particular in the fields of infectious diseases [ 1 , 5 ] and cancer, where large proficiency panels have been conducted both in Europe and the United States within the remit of the Association for Cancer Immunotherapy Immunoguiding Programme (CIMT-CIP) [ 6 ] and the Cancer Immunotherapy Consortium of the Cancer Research Institute (CIC-CRI) [ 7 ], respectively. Such panels have investigated factors that influence the frequency of responses detected by ex vivo IFNγ ELISPOT [ 6 , 8 , 9 ] and multimer staining [ 10 , 11 ].…”

Vaccine against autoimmune disease: antigen-specific immunotherapy

“…Olson and colleagues formally tested conditions for shipping fresh blood to be used in T cell assays at central labs and found that maintaining specimen temperature above 22 °C or preferably near 30 °C was optimal [75]. Filbert et al defined methods for cryopreservation that yielded optimal T cell responses in subsequent ELISPOT analysis [76]. And perhaps, most importantly, the reporting of immune readouts has been addressed by the publication of minimal information about T cell assays (MIATA) in an effort to harmonize assays between laboratories [77••].…”