Abstract
Introduction
Monocytes, a type of circulating immune cell, play a pivotal role in the innate immune response. Their functions, including phagocytosis, wound healing, cytokine secretion, and inflammation, are crucial for resolving many pathologies but may also contribute to inflammatory diseases. Classical monocytes are the most abundant monocyte subset and specialize in phagocytosis and inflammatory cytokine secretion. Cryopreservation is commonly used to enable the collection and storage of large numbers of classical monocytes, usually within peripheral blood mononuclear cells (PBMCs) from a single blood sample. This technique allows for comparability of samples across multiple experiments, particularly during optimization. However, cryopreservation protocols are often overlooked in study optimization efforts. This thesis aimed to optimize the isolation and analysis of classical monocytes from cryopreserved PBMCs, focusing on maintaining cell viability, yield, and the accurate measurement of relevant classical monocyte functions and activation markers.
Methods
PBMCs were isolated from healthy volunteers using density gradient centrifugation and cryopreserved. Classical monocytes were isolated from thawed samples using negative magnetic bead selection. These cells were phenotyped using spectral flow cytometry or stimulated with either ionomycin or LPS for functional assays. A reactive oxygen species (ROS) assay was used as a standard marker of monocyte function and included the use of a fluorogenic probe, CellROX Green, that could be analyzed using flow cytometry. Antibodies for common monocyte activation markers CD11b, CD11c, and HLA-DR were also included to assess activation status after in vitro stimulation.
Results
Including a resting step after thawing cryopreserved PBMCs did not improve classical monocyte yield or phenotype, compared to fresh samples. Density gradient centrifugation to isolate PBMCs for cryopreservation, significantly increased the incidence of monocyte-platelet aggregates. The impact of these aggregates on the chosen functional assays remains unknown but is a potential avenue for future research. A precise staining environment was required for CellROX green to accurately detect levels of ROS production by both unstimulated and ionomycin-stimulated monocytes. Additionally, CellROX green can be stained alongside multiple other phenotyping antibodies to quantify ROS production, monocyte phenotype, and activation, simultaneously. Varying LPS concentrations did not induce ROS production detected by CellROX green at either 30 minutes or 4 hours post-stimulation.
Conclusion
The methods resulting from this thesis provide a solid foundation for continued investigation. Further research could explore whether monocyte-platelet aggregates or elements of the cryopreservation process may affect the function and phenotype of monocytes stored in the Wellington Cardiovascular Research Groups’ biobank for use in clinical studies.