Development of an abbreviated protocol for the polarization and characterization of therapeutic inflammation-resolving monocytes/macrophages

ConclusionPeripheral blood MΦs are isolated by density centrifugation and CD14 magnetic beads, then treated with in-house cytokine cocktail or small molecule inhibitors for 24-48h. Polarized MΦs are co-cultured with late stage osteoarthritic explants to test phenotypic stability. MΦs are characterized through: flow cytometry (surface markers, viability, dextran endocytosis), qRT-PCR and/or immunoassay (inflammatory/chemotactic/catabolic mediators), and T cell proliferation. Treatment phenotype is compared against 7-day treatment with M-CSF and against naïve 48h MΦs.48h polarized MΦs demonstrate a similar phenotypic profile to 7-day polarized MΦs. Gene expression of anti-inflammatory IL-10 is upregulated in both groups relative to naïve controls. Surface expression of scavenger receptors CD163 and CD206, markers of an inflammation-suppressive phenotype, are higher in both the 48h and 7d groups relative to control. Pro-inflammatory markers CD86 and HLA-DR are not highly expressed in either group. 48h polarized MΦs maintain a similar surface marker profile after co-culture with osteoarthritic explant tissue for up to 7 days.Preliminary results demonstrate that the 48h polarization protocol yields a similar phenotypic profile to 7d polarized MΦs. With further functional assay and protocol optimization using small molecule agents, we aim to develop an efficient, well-characterized inflammation suppressive MΦ phenotype that can be easily translated for clinical use.
Source: Cytotherapy - Category: Cytology Source Type: research