077 PBMC gene expression profiles: Impact of sample handling

Background: Previously, we showed that human peripheral blood mononuclear cells (PBMCs) that were cryopreserved, stored for 14 months at: (1) −150 °C; (2) −150 °C except temperature cycled for 104 h; and (3) −80 °C and then thawed had numerous genes that were differentially regulated compared to freshly isolated cells. Study Design and Methods: We have expanded our initial report to include a 6.5 year time point with aliquots from the same 10 donors in order to determine the effect of longer storage time on gene expression from these samples. We also collected another 10 donors (4 donors were identical from the 1st collection). Identical processing and storage methodology was done as previously, except we decreased the initial storage interval to 3 months and cultured aliquots for 24 h to more precisely determine when gene expression changes occur and if they are reversible, respectively. We also performed flow cytometry and viability analysis on samples to monitor changes in cell phenotype and apoptosis/necrosis that occurred during sample handling. Significantly modulated genes ( p  2∣-fold change) were selected based on ANVOVA with contrast analysis for comparison of difference storage conditions and storage time. Genes that correlated with cell viability values were selected based on r 2  > 0.3 ( p 2-fold difference in the gene expression value. Results: Gene expression analysis indicated little difference at 3 months between fresh and the 3 storage conditions which was consistent with no significant differences in cell viability between fresh and the 3 storage conditions. At 6 years nearly 600 genes were differentially modulated in the −80 °C stored samples and a couple of hundred genes were altered in the LN2 and LN2-cycled condition; the mean percent of recovered viable cells was 98% for fresh, 78% LN2, 68% LN2-cycled and 9% for −80 °C. Culturing of fresh cells and stored cells consistently modulated a set of the same 1800 genes that where highly enriched in various aspects of immune activation, most likely the result of stimulation by serum. To better understand the biological pathways involved in poor cell recovery following cryopreservation, we correlated cell viability with gene expression levels across all storage conditions and times. In uncultured samples, the percentage of dead cells correlated with genes significantly enriched in apoptosis, MAP kinase, c-jun, heat shock and stress response pathways. When these same samples were cultured for 24 h, the percentage of dead cells correlated with genes significantly for enriched cytokine, inflammation and immune response pathways. Our presentation will also discuss and relate our current findings to how storage conditions and time in storage also influence cell subset frequency, apoptosis level and necrosis level and could lead to candidate biomarkers of quality for cryopreserved human PBMC samples. Conclusion: Expression profiling identified genes involved in apoptosis and stress–response pathways that are associated with poor cell viability caused by suboptimal handling; and provides candidate pathways to target with inhibitors of cell death to improve cellular recovery and function. Culturing of low quality samples results in increased expression of cytokines and immune response genes which may severely skew the interpretation of functional assays. Source of funding: Funded by NCI Contract No. HHSN261200800001E. Conflict of interest: None declared. rlempicki@mail.nih.gov