MONOCYTE-DERIVED DENDRITIC CELLS ISOLATION AND PHENOTYPIC CHARACTERIZATION

Mohamed Farag

doi:10.21608/ajps.2020.118369

Abstract

Dendritic cell (DC) is the most powerful inducers and regulators of immune responses, responsible for interaction within the immune system. The ability of DC to induce or regulate/suppress immune responses led to attention in their immunotherapeutic use in various disease types. The aim of the presented study was to generate in vitro CD14+ enriched peripheral blood monocytes (PBMCs) following the culture with human monocyte-derived dendritic cell serum-free differentiation media over ten days with phenotypical analysis and morphological identification for functional studies. In vitro, peripheral blood of human donors samples were collected for the purification of blood CD14+ monocytes that represent the most common origin for DC precursors based on ficoll density gradient separation and human monocyte-derived dendritic cell enriched differentiation medium. After PBMCs isolation, the cell viability, cell yield were determined, the monocyte-derived dendritic cell surface marker expression was detected by flow cytometric analysis after staining with specific fluorescence-conjugated monoclonal antibodies. in vitro culture and differentiation of human monocytes into DCs and their subsequent maturation into mature DC constitute a critical first step for different downstream analysis ranging from an understanding of both human DC biology and function to their use in clinical diagnostics and therapeutic design for different disease.

Highlights

Dendritic cells (DCs) are the most professional antigen-presenting cells (APC) of the mammalian immune system that represent a key mediator of both innate and adaptive immune responses (Steinman, 2012)
The main objective of this study is to generate in vitro immature dendritic cells from CD14+ peripheral blood monocytes (PBMCs) by means of a differentiation and maturation process induced by exogenous stimuli (Recombinant Human interleukin 4 (IL-4), Recombinant Human granulocyte monocyte colony-stimulating factor (GM-CSF) and Recombinant Human tumor necrosis factor α (TNF-α) ) with morphological determination and phenotypic identification of PBMC-derived DCs following culturing in a defined period which represent a crucial step for studying the biology of DCs, their roles in immune responses, and their potential use as immunotherapeutic tool against both malignant cell antigens and infective microorganisms
Some of the assumptions made about human DC are turned from small animal experiments that have not been confirmed in man and may not necessarily apply (Ju, 2010). monocyte-derived Dendritic Cell (moDC) are a subset of DCs widely used in immunological studies as a convenient and easy approach after isolation of mononuclear cells directly from circulation (Fol et al, 2016)

Summary

Introduction

Dendritic cells (DCs) are the most professional antigen-presenting cells (APC) of the mammalian immune system that represent a key mediator of both innate and adaptive immune responses (Steinman, 2012). Due to their abilities to pathogen recognition, capturing and processing machinery, migratory capacity and cytokines production that promotes pathogen-specific effector T cell differentiation and activation, growing interest in their use in clinical approaches has been observed. Immature DCs express specific pattern recognition receptors that serve as expression markers and allow for the capture and processing of foreign antigens following infection (Harman et al, 2013). Cytokines produced by DCs can promote the differentiation of CD4+ T helper cells as part of immune activation (Bol et al, 2019)

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