title: 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.
authors.affiliation: III. Medical Department, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.
authors.forename: Helene
authors.initials: H
authors.lastname: Filbert
authors.forename: Sebastian
authors.initials: S
authors.lastname: Attig
authors.forename: Nicole
authors.initials: N
authors.lastname: Bidmon
authors.forename: Bernhard Y
authors.initials: BY
authors.lastname: Renard
authors.forename: Sylvia
authors.initials: S
authors.lastname: Janetzki
authors.forename: Ugur
authors.initials: U
authors.lastname: Sahin
authors.forename: Marij J P
authors.initials: MJ
authors.lastname: Welters
authors.forename: Christian
authors.initials: C
authors.lastname: Ottensmeier
authors.forename: Sjoerd H
authors.initials: SH
authors.lastname: van der Burg
authors.forename: Cécile
authors.initials: C
authors.lastname: Gouttefangeas
authors.forename: Cedrik M
authors.initials: CM
authors.lastname: Britten
mesh_terms: D000818:Animals; D002417:Cattle; D002470:Cell Survival; D016895:Culture Media, Serum-Free / Q000737:chemistry*; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods* / Q000592:standards; D018984:Epitopes, T-Lymphocyte / Q000032:analysis / Q000276:immunology; D005615:Freezing; D015789:HLA-A2 Antigen / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000737:chemistry* / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D013601:T-Lymphocytes / Q000737:chemistry / Q000276:immunology
chemical_list: D016895:Culture Media, Serum-Free; D018984:Epitopes, T-Lymphocyte; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D010446:Peptide Fragments; D007371:Interferon-gamma