In vitro culture of human monocytederived macrophages with regards to M1/M2 polarization
Examensarbete för masterexamen
Biotechnology (MPBIO), MSc
Wound healing is a complex process where macrophages are highly involved, both by clearing the tissue from bacteria and debris, but also by regulating the inflammation and healing outcome by production of various cell mediators. A characteristic feature of macrophages is their ability to display a spectrum of different phenotypes. They can roughly be divided into two extreme phenotypes; M1 macrophages which produce pro-inflammatory cytokines and metalloproteinases and is strongly antimicrobial and M2 macrophages which produce anti-inflammatory cytokines, resolving inflammation and promoting tissue repair. Although both phenotypes are key in wound healing, an imbalance of M1 macrophage domination can result in nonhealing ulcers. At Mölnlycke Health Care effort is being paid at developing wound care products that could enhance the healing. In order to develop such concepts, an in vitro inflammatory model with M1/M2 macrophages could be utilized. The aim of the thesis is to identify and study the relation of cell mediators and markers related to M1/M2 polarization in cultured human monocyte-derived macrophages and also to create a theoretical link of relevance of chosen markers to data in murine models and the human diabetic ulcer. Monocytes were isolated from buffy coats, obtained from healthy blood donors. To induce M1 and M2 macrophages, cells were stimulated with LPS and IFN- (M1) or IL-4 (M2) for 6, 24, 48 or 72 hours prior to analysis. M1 cells where further reversibly polarized with IL-4 after 48 hours in order to switch phenotype from M1 to M2 cells. Secreted markers of M1/M2 macrophages were studied with ELISA. Production of TNF- and IL-1 indicated a M1 phenotype and CCL18 production indicated a M2 phenotype. Production of IL-10 which is a M2 phenotype marker was produced by M1-stimulated macrophages. No production of IL-23 and IL-1ra was seen. Further, no significant CCL18 production was seen after reversible polarization, indicating that M1 macrophages did not switch phenotype to M2 macrophages. Different assay setups of lactate and reactive oxygen species production (M1 markers) did not display any significant results in this study.
Annan humaniora , Grundläggande vetenskaper , Hållbar utveckling , Innovation och entreprenörskap (nyttiggörande) , Livsvetenskaper , Other Humanities , Basic Sciences , Sustainable Development , Innovation & Entrepreneurship , Life Science