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Peripheral Blood Mononuclear Cells

Charlotte R. Kleiveland

Abstract Numerous cell types are involved in maintenance of the intestinal tissue. However, the main players are cells of the epithelial lining and the immune system. Human peripheral blood mononuclear cells (PBMCs) are used to investigate the effect of food bioactives on various immune cells. These cells are easily isolated from blood of healthy donors or buffy coats (leukocyte concentrates, a by-product from hospital Blood Banks in the manufacturing of red blood cell and thrombocyte concentrates from anti-coagulated whole blood). PBMCs have a different composition, phenotype and activation status than cells found in intestinal tissue. However, this is a useful test system for investigation of immune modulatory effects of food bioactive compounds. Methods for the isolation of PBMCs and how they are used to investigate effects of bioactive components are discussed in this chapter.

Keywords Peripheral blood mononuclear cells (PBMCs) • Mitogenic lectins • T cells • Cytokines • Surface markers

Origin

Human peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood and identified as any blood cell with a round nucleus (i.e. lymphocytes, monocytes, natural killer cells (NK cells) or dendritic cells). The cell fraction corresponding to red blood cells and granulocytes (neutrophils, basophils and eosinophils) is removed from whole blood by density gradient centrifugation. A gradient medium with a density of 1.077 g/ml separates whole blood into two fractions; PBMCs makes up the population of cells that remain in the low density fraction (upper fraction), whilst red blood cells and PMNs have a higher density and are found in the lower fraction (Fig. 15.1).

Fig. 15.1 Schematic illustration of how to prepare the density gradient for isolation of PBMCs from blood (a, b) and where in the gradient the PBMCs (grey layer) are found after centrifugation (c, d)

PBMCs include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. In humans, the frequencies of these populations vary across individuals, but typically, lymphocytes are in the range of 70–90 %, monocytes from 10 to 20 %, while dendritic cells are rare, accounting for only 1–2 %. The frequencies of cell types within the lymphocyte population include 70–85 % CD3+ T cells, 5–10 % B cells, and 5–20 % NK cells. The CD3+ lymphocytes are composed of CD4+ and CD8+ T cells, roughly in a 2:1 ratio. After activation the CD4+ T cell subset may develop into diverse effector cell subsets, including Th1, Th2, Th17, Th9, Th22, follicular helper (Tfh) cells and different types of regulatory cells (Akdis et al. 2012; Crotty 2011; Tan and Gery 2012; Sakaguchi et al. 2008). The CD4+ helper T cells are essential mediators of immune homeostasis and inflammation (Hirahara et al. 2013).

 
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