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.

Fig. 1

Overview of experiments. The experimental design of the study is depicted as a flow chart indicating the starting sample specimens and freezing media applied (two boxes in the top). Performed experiments were a either conducted in a proficiency panel with 31 participating laboratories comparing cells frozen with three different media or b in the central laboratory comparing cells frozen with seven different media. The two boxes in the center of the flow chart indicate the number of investigators that did the experiments, the number of assay protocols that were used and the number of replicates for each experiment. The box at the bottom indicates the experimental readouts that were made in all experiments and are reported in the “Results” section

Fig. 2

Viability, recovery and resting loss in the proficiency panel. To illustrate the distribution of recovered cells, viability and resting loss for the different freezing conditions box plots were used. The rectangle shows the interquartile range ranging from the first quartile (the 25th percentile) to the third quartile (the 75th percentile). The whiskers point at the minimum and maximum value unless the distance from the minimum value to the first quartile is more than 1.5 times the inter-quartile range (IQR). In that case, the whisker extends out to the smallest value within 1.5 times the IQR from the first quartile. The circles indicate outliers, which are smaller or larger than the whiskers. The lines inside the rectangle show the median. The box plots show the results obtained for all media and stratified by freezing medium conditions A (90 % human AB serum + 10 % DMSO), B [CryoMaxx II (PAA)] and C (10 % human serum albumin + 10 % DMSO + 80 % RPMI). a Viability of cells directly after thawing. Statistical testing (unpaired t test) was performed (A vs. B: p < 0.0001; A vs. C: p = 0.0015). b Recovery of viable cells per vial directly after thawing. Statistical testing (unpaired t test) was performed (A vs. B: p = 0.01; A vs. C: p = 0.046). c Cell loss during resting. Statistical testing (unpaired t test) was performed (A vs. B: p = 0.068; A vs. C: p = 0.0345)

Fig. 3

Cell viability and recovery after thawing for seven different freezing media and three donors tested in one center assay. The figure shows results obtained with cells from donors 1–3. The filled symbols show results obtained immediately after thawing. Open diamonds show results after resting of cells, prior to testing. a Viability of cells (mean result of triplicate at two independent experiments). The quality of cells after thawing and resting was high (median viability 95 %). b Recovery of cells (mean result of triplicates from two independent experiments) is indicated as percentage of viable cells that was recovered from each thawed vial relative to the number of cells that were originally filled in each vial

Fig. 4

Immunologic function of cells in one center assay. Results are compiled from two independent experiments with cells frozen with seven different freezing media and expressed as mean spot numbers for each of the three donors (donors 1–3) tested. Antigen-specific T-cell responses are indicated as spots per 100,000 PBMCs seeded per well. a Mean background spot production in the medium control wells. b Mean number of antigen-specific spots against the CMV peptide for all three CMV-reactive donors. c Mean number of antigen-specific spots against the FLU peptide for the two influenza-reactive donors. Triangles D1, circles D2, squares D3