Umbilical Cord Blood-Derived Monocytes as A Reliable Source of Functional Macrophages for Biomedical Research

Document Type : Original Article


1 Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

2 Department of Applied Cell Sciences, Faculty of Basic Sciences and Advanced Medical Technologies, Royan Institute, ACECR, Tehran, Iran

3 R & D Department, Royan Stem Cell Technology Co, Tehran, Iran

4 Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5 Physiology Research Center, Kashan University of Medical Sciences, Kashan, Iran

6 R and D Department, Royan Stem Cell Technology Co, Tehran, Iran

7 Department of Laboratory Medicine, Division of Pathology, Karolinska Institute, Stockholm, Sweden

8 Department of Pathology and Cancer Diagnostic, Karolinska University Hospital, 141 83 Stockholm, Sweden

9 Experimental Cancer Medicine, Institution for Laboratory Medicine, and Karolinska University Hospital, Karolinska Institute, Stockholm, Sweden


Objective: Macrophages are multifunctional immune cells widely used in immunological research. While autologous
macrophages have been widely used in several biomedical applications, allogeneic macrophages have also
demonstrated similar or even superior therapeutic potential. The umbilical cord blood (UCB) is a well-described
source of abundant allogenic monocytes and macrophages that is easy to collect and can be processed without
invasive methods. Current monocyte isolation procedures frequently result in heterogenous cell products, with limited
yields, activated cells, and high cost. This study outlines a simple isolation method that results in high yields and pure
monocytes with the potential to differentiate into functional macrophages.
Materials and Methods: In the experimental study, we describe a simple and efficient protocol to isolate highpurity
monocytes. After collection of human UCB samples, we used a gradient-based procedure composed of three
consecutive gradient steps: i. Hydroxyethyl starch-based erythrocytes sedimentation, followed by ii. Mononuclear
cells (MNCs) isolation by Ficoll-Hypaque gradient, and iii. Separation of monocytes from lymphocytes by a slight
hyperosmolar Percoll gradient (0.573 g/ml). Then the differentiation potential of isolated monocytes to pro- and antiinflammatory
macrophages were evaluated in the presence of granulocyte colony-stimulating factor (GM-CSF) and
macrophage CSF (M-CSF), respectively. The macrophages were functionally characterized as well.
Results: A high yield of monocytes after isolation (25 to 50 million) with a high purity (>95%) could be obtained from
every 100-150 ml UCB. Isolated monocytes were defined based on their phenotype and surface markers expression
pattern. Moreover, they possess the ability to differentiate into pro- or anti-inflammatory macrophages with specific
phenotypes, gene/surface protein markers, cytokine secretion patterns, T-cell interactions, and phagocytosis activity.
Conclusion: Here we describe a simple and reproducible procedure for isolation of pure monocytes from UCB, which
could be utilized to provide functional macrophages as a reliable and feasible source of allogenic macrophages for
biomedical research.


Main Subjects

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