Optimization of Isolation Processing of Monocytes from Peripheral Blood Mononuclear Cells and Differentiation into Dendritic Cells

Heo, Yoon Jeong; Park, Mi Young; Kim, Hyung Hoi; Park, You Soo; Son, Jeong Hwa

Abstract

BACKGROUND
Rescently, the immunotherapy against infectious disease and cancer is being actively studied, and high yield of pure CD14+ monocytes is a key of concern. In this study, we optimized the percoll gradient method to increase the purity and yield of monocytes from peripheral mononuclear cells.
METHODS
We separated mononuclear cells (MNC) from healthy donors, and monocytes from MNC were separated with the various density of percoll gradient. After centrifugation, we determined the most efficient density of the percoll gradient to get the highest yield and purity of monocytes. We also obtained monocytes by the plastic absorption method. Monocytes were differentiated into dendritic cells (DC) and the efficacy of differentiation to DC is confirmed and compared by morphological characteristics and using a flowcytometer.
RESULTS
The purity of monocytes was 51.3+/-8.3% by the 35% percoll gradient method and the purity was improved to 82.9+/-4.2% with 100% of yield by repeating the same method. Therefore, the yield of mature dendritic cells was 3.6+/-0.9% of the total input MNC by the 35% percoll repetition method, which was 1.7 times higher than the plastic adherent method (2.1+/-0.5%).
CONCLUSION
This study shows a cost-effective method to isolate CD14+ monocytes and these cells demonstrate high differentiation rate to DC. This process will be valuable for obtaining a sufficient number of DC.